Bisphenol A exposure Alters Placentation and Causes Preeclampsia-like Features in Pregnant Mice Involved in Reprogramming of DNA Methylation of WNT-2

Abstract

Bisphenol A (BPA) exposure is shown to be positively associated with increased risk of preeclampsia; However, there is no experimental research supporting their causal relationship, and the underlying mechanisms by which BPA exposure causes preeclampsia remains unknown. Here, we investigated whether BPA exposure causes preeclampsia in pregnant mice, and the underlying mechanisms. Using a mouse model, we found that pregnant mice exposed to BPA exhibited preeclampsia-like features at the minimum effective dose of 4 μmol/L. Preeclampsia-like features included hypertension, disrupted circulating and placental angiogenesis biomarkers of Flt-1 and PlGF, and atrophy glomerular. These preeclampsia features were correlated with increased retention of smooth muscle cells and the reduced vessel areas at the junctional zone of placenta, associated with decreased invading trophoblast cells and disrupted expressions of invasion and epigenetic modification related genes including increased TIMP-1, TIMP-2, DNA methylation transferase-1 (DNMT-1), and decreased MMP-2, MMP-9, β-catenin, and WNT-2. Remarkably, BPA exposure-reduced expression of WNT-2 was correlated with the increased DNA methylation in its promoter region. In HTR-8/SVneo cells, BPA exposure impeded its interaction with HUVEC, and down-regulated WNT-2 expression and elevated its DNA methylation, which is consistent with in vivo observations. Inhibiting DNMTs in HTR-8/SVneo cells resulted in reduced DNA methylation and increased expression of WNT-2. Take together, these data demonstrate that BPA exposure altered trophoblast cell invasion and caused abnormal placental vessel remodeling, leading to the development of preeclampsia-like features in pregnant mice, which involves the epigenetic reprogramming and down-regulation of WNT-2 mediated by DNMT-1.

Introduction

Preeclampsia is a pregnancy-specific disease that is characterized by new-onset of hypertension and urinary protein after the 20^th week of gestation and remains one of the major cause of adverse pregnancy and birth outcomes worldwide¹. Despite the severe consequences of preeclampsia on maternal and fetal health, the pathophysiological mechanisms are unclear. However, inadequate trophoblastic invasion and remodeling of maternal spiral artery early in pregnancy are widely considered to play vital roles in preeclampsia¹.

During early pregnancy in human, trophoblasts must anchor and invade maternal decidualized endometrium. The invasive trophoblasts destroy the maternal spiral arterial wall, replacing the endothelial cells to generate low-resistance, large-diameter vessels that promote uteroplacental blood flow to sustain fetal growth. Poor endovascular trophoblast cell invasion diminishes the remodeling and diameter of spiral arteries including retention of smooth muscle, which increases the vessel’s contractility and vasoconstriction in preeclampsia. It is well established that uteroplacental cytoactive factors such as matrix metalloproteinases (MMPs), tissue inhibitor of MMPs (TIMPs), canonical Wnt family member 2/beta-catenin (WNT-2/β-catenin) signaling, and imbalanced pro-angiogenic/anti-angiogenic factors of soluble fms related tyrosine kinase 1 and placenta growth factor (sFlt-1/PlGF) are associated with decreased vascular remodeling and trophoblast invasion of the myometrium.

Among all risk factors of preeclampsia, environmental factors are considered to be as equally important as genetic factors. Previous studies indicate that maternal exposure to the environmental chemical BPA may be an important risk factor for abnormal placentation^2–4 and subsequent pregnancy complications including preeclampsia^{5, 6}.

BPA, known as an endocrine-disrupting chemical (EDC), is one of the highest volume man-made chemicals and widely used synthetic plasticizers. Humans are widely exposed to BPA primarily by leaching from food and beverage containers through diet⁷. BPA is detectable in urine, serum, and placenta tissue of pregnant women⁷. Epidemiology studies reported the positive association between maternal exposure of BPA and the risk of preeclampsia^{5, 6,8}. BPA exposure affected placental morphology and angiogenesis during early pregnancy in mice³, which might involve WNT-2 signaling pathway³ as BPA exposed mice placentas exhibited altered placental global DNA methylation^{2, 9}. Epigenetic regulation is vital during placental development¹⁰ and abnormal epigenetic regulation in placenta is associated with preeclampsia¹⁰. Although increasing evidence suggested a positive association between BPA exposure and preeclampsia^{2, 3, 5, 6,8}, there is no supporting evidence for a causal relationship between BPA exposure and preeclampsia.

The objective of the present study is to establish a causal relationship between maternal BPA exposure and the risk of preeclampsia, and to shed light on the underlying mechanisms using an in vivo mice model and an in vitro human cell model. We hypothesized that BPA exposure induces preeclampsia-like features in pregnant mice and the underlying mechanisms involves abnormal placental vessel remodeling, altered trophoblast cell invasion and relevant signaling pathways. To test our hypothesis, mice were exposed to BPA starting at embryo day (E)-7. Preeclampsia-like features were characterized with a combination of observations including systolic blood pressure (SBP), urinary protein level, renal and placental morphology, and circulating/placental Flt-1/PlGF ratio. Using both mice placenta tissues and a human cytotrophoblast cell line, HTR-8/SVneo, we examined the placental vessel remodeling and trophoblast invasion related signaling pathways including matrix metalloproteinases (MMPs) and WNT-2 under BPA exposure. We further explored the role of methylation in WNT-2 expression under BPA exposures.

Methods

The authors declare that all the supporting data are available within the article and its online data supplement. The detailed description of material and methods are provided online as supplementary data. Briefly, pregnant mice were exposed to BPA through drinking water from embryonic day (E)-7 to E17. Urinary BPA concentrations were measured by liquid chromatography-mass spectrometry (LC-MS). Preeclampsia was characterized by a combination of measurements including blood pressure, proteinuria, circulating and placental biomarkers of Flt-1 and PlGF, and the pathological changes of the placenta and kidney. PCR/Western blot/ELISA/immunostaining were used to analyze various trophoblast invasion related genes and protein expressions of MMP-2, MMP-9, TIMP-1, TIMP-2, WNT-2 and β-catenin. PCR-Array was used to profile gene expression of epigenetic chromatin modification enzymes. Bisulfite sequencing was used to measure DNA methylation of WNT-2 and β-catenin. Using human cells, we further examined the impacts of BPA exposure on cytotrophoblast invasion, invasion relevant WNT signaling, and epigenetic regulation.

Statistical analysis

Data were represented as Mean ± SEM. The differences between control and treatment groups were examined using Student’s t test or ANOVA analysis. Statistical analysis was performed using Prism 5 software. Comparisons were considered statistically significant if P<0.05.

Results

BPA exposure induced preeclampsia-like features in pregnant mice

BPA exposure levels were confirmed by 24h-urinary BPA quantification (Fig1.A). 24h total urinary BPA levels rose progressively with increasing exposure doses of BPA. The elevated urinary BPA levels in mice exposed to BPA at doses higher or equal to 4μmol/L were statistically significant compared with no BPA exposed control mice (P<0.05). BPA exposures significantly elevated SBP in pregnant mice at levels of equal to or greater than 4μmol/L (4 μmol/L BPA vs control: 131.5 ± 7.66 mmHg vs 104.9 ± 3.78 mmHg; P<0.01) (Fig1.B), but non-pregnant mice were not affected by BPA exposure at a level of 400μmol/ L (P=0.36) (Fig1.B). 24h total urine protein at E17 in BPA exposed groups showed no significant difference from that in the control group (Fig1.C). A dosage of 4μmol/ L of BPA was regarded as the minimum effective dosage for elevated SBP in pregnant mice and was chosen for further analysis in this study. Histopathological analysis of renal tissues collected from control and BPA (4μmol/L)-treated pregnant mice was performed. In the control group, there was no significant histological changes in renal tissue. While in the BPA exposure group, atrophy glomerular, narrowed glomerular cystic cavity, and glomerular capillary loops occuping the whole glomerulus were showed in varying degrees. Placental and circulating Flt-1 (Fig1.E; G) levels and Flt-1/PlGF ratio (Fig1.F; I) in response to BPA exposure (4 μmol/L) were significantly elevated compared with no-BPA-exposed control mice, although changes of PlGF levels were not statistically significant. Maternal body weight, litter size, mean fetal weight, mean placental weight and placenta/fetus weight ratio did not show significant variation in response to BPA (P>0.05) (Fig.1 J; K; L; M; N). Preterm birth was not observed in either the BPA exposed or control groups.

Fig1. BPA-exposed pregnant mice exhibited preeclampsia-like features and impaired placental angiogenesis.

(A) 24h total urinary free BPA levels at E17 rose with increased exposure levels of BPA. N=3. (B) SBP at E16 was elevated by BPA exposure. N=8. (C) 24h total urinary protein level at E17 was not affected. N=5. (D) Renal at E17 shows constricted glomerular (H&E; left) and thicken basement membrane (PAS; right). (Scale bar: 20μm). (E) BPA exposure (4 μmol/L) altered protein expressions of Flt and PlGF in mice placenta at E17 (N=3). (F) Gray values were quantified. N=3. (G) (H) BPA exposure (4 μmol/L) altered serum levels of sFlt-1 and PlGF at E17, quantified by ELISA. N=3. (I) sFlt-1/ PlGF ratio was increased by BPA exposure. N=3. (J) (K) (L) (M) (N) BPA exposure (4 μmol/L) did not alter maternal weight, litter size, mean fetal weight, mean placental weight or placental/fetal weight ratio. N=8. (O) Placenta at E17 shows abnormal lesions in placental labyrinth (arrow heads) (H&E; left) and collagen deposition (arrow heads) (MTC; right). (Scale bar: 50μm). (P) Implantation site at E13 shows elevated retention of smooth muscle cells in decidua vessels immunostained for α-SMA. (Q) Vessel area was measured in placental cross section at E13 and reduced by BPA exposure. N=5. (R) BPA exposure did not affect cell proliferation in placenta at E13 embryo sections showed by Ki67 (Scale bar: 100μmol/L). (S) Immunohistochemical scores of Ki67 were quantified.

Quantitative data are presented as mean ± SEM. Students t test was performed to determine the statistical significance of two comparisons. *P<0.05, **P<0.01, ***P<0.001.

Impaired placental structure and reduced placental vessel remodeling

Compared with controls, placenta from BPA exposed mice exhibited abnormal lesions shown by H&E staining (Fig1.O.H&E) and was confirmed to be fibrous matrix deposition by MTC staining (Fig1.O.MTC). α-SMA-labeled vascular smooth muscle cells (VSMC) retained and pan-CK-labeled trophoblast cells were decreased in vessels at maternal side of junctional zone(Fig1.P), and vessel area was significantly reduced (68.26 ± 1.20 vs 164.4 ± 28 μm², P=0.01) (Fig1.Q). Placental cell proliferation was not different between control and 4μmol/L BPA-treated mice demonstrated by immunostaining of a cell proliferation marker, Ki67 (Fig.1.R; S).

BPA exposure altered MMP s and WNT-2 signaling pathways

In 4μmol/L-BPA-exposed mice, BPA disrupted trophoblast invasion related gene profile, including increased transcripts of invasion-inhibited genes, TIMP-1 and TIMP-2 (Fig2.A), and decreased transcripts of invasion-stimulated genes, MMP-2, MMP-9 (Fig2.A), WNT-2 (Fig2.B) and β-catenin (Fig2.C), as well as WNT-2 and β-catenin proteins (Fig2.D) in mice placental extracts compared to no-BPA-exposed mice, indicating a role of BPA in suppressing trophoblast cell invasion.

Fig2. BPA altered trophoblast cell invasion related signaling pathways, DNMT-1 expression, and DNA methylation of WNT-2 gene in mice placenta at E13.

(A) BPA exposure (4μmol/L) altered MMP -2, MMP -9, TIMP-1, TIMP-2 gene expressions in placenta at E13. N=3. (B) (C) BPA exposure reduced gene expressions of WNT-2 and β-catenin in placenta at E13. N=3. (D) Reduced protein expressions of WNT-2 and β-catenin in placenta at E13. N=3. (E) (F) Altered protein expressions of DNMTs and TETs in placenta at E13. N=3. (G) Altered expressions of 5-mc (left) and 5-hmc (right) immunostaining in placenta at E13 (Scale bar, bottom: 200μm, top right: 100μm). (H) (I) Immunohistochemical scores of 5-mc and 5-hmc were quantified. N=5. (J) BPA elevated total 5-mc levels in placenta at E13, quantified by ELISA, N=5. (K) BPA elevated DNA methylation of mouse WNT-2 gene in promoter of 22CpG sites. (Each circle shows the average methylation of an individual CpG site; open circle=0% methylation, the last circle of average =percent of total methylation per sample), N=5. (L) Total percent of DNA methylation (%) of CpG site of WNT-2 gene was increased by BPA exposure. N=5.

Quantitative data are presented as mean ± SEM. Students t test was performed to determine the statistical significance of two comparisons. *P<0.05, **P<0.01, ***P<0.001.

We then examined whether BPA exposure regulated these signaling pathways through epigenetic mechanisms. For initial screening using PCR Array analysis of 84 genes related to epigenetic modification in mice placenta, we found that BPA exposure up-regulated 8 genes and down-regulated 2 genes (≥ two fold) (Table1), involving histone methylation and acetylation, protein phosphorylation and DNA methylation. Among the 10 regulated genes, the change of DNMT-1 was the most remarkable one (fold changes=2.36) (Table1). We further confirmed that BPA exposure significantly elevated protein expression of DNMT-1 (Fig2.E) in placenta, while DNMT-3a, DNMT-3b (Fig2.E), hydroxymethylation transferases (TET-1, TET-2 and TET-3) (Fig2.F), and 5-hmc (Fig2.F; H) were not affected. Further, elevated level of 5-mc (Fig2.F; I; J) indicated an elevated methylation level in mice placenta under BPA exposure.

Table1.

Differently expressed genes in mice placenta at E13

m-RNA	Fold changes*
DNMT-1	2.36
Esco1	2.03
Kat2b	2.07
Kdm5b	2.17
Prmt7	2.06
Setd1a	2.06
Setd6	2.06
Setd8	2.14
Gusb	2.01
Aurkc	0.05
Ciita	0.42

Placental gene expression at E13, qualitied by real-time PCR array, N=2.

^*Mean was compared between BPA exposure (4 μmol/L) and control group.

After it was confirmed that BPA exposure regulated methylation enzymes in mice placenta, we examined whether WNT-2 expression was affected by BPA exposure through methylation reprogramming. The average DNA methylation level of WNT-2 was elevated about 50% in the BPA exposed group compared with controls (15.12% ± 1.68 vs 7.88% ± 2.35, P=0.04) (Fig2.K; L) and the main affected CpG sites were at CpG1, CpG2, CpG3 and CpG4 (Fig2.K). The DNA methylation of β-catenin was not affected by BPA exposure (Fig s2.A; B. Supplementary file).

BPA treatments and WNT-2 signaling in HTR-8/SVneo cells

BPA exposure decreased cell viability of HTR-8/SVneo cells at 100μmol/L showed by CCK-8 test (100μmol/L BPA vs control: 14.63%±2.63% vs 99.98% ± 21.87%, P=0.02) (Fig3. A).

Fig3. BPA decreased WNT-2 and β-catenin expressions and this down-regulation was mediated through up-regulation of DNA methylation in HTR-8/SVneo cells; and BPA impaired HTR-8/SVneo / endothelial cell interaction.

(A) HTR-8/SVneo cell viability following BPA treatments was detected using CCK-8. N=3. (B) BPA exposure altered protein expression of DNMTs in HTR-8/SVneo cells. (C) Gray value were qualified, N=3. (D) BPA elevated DNA methylation of 23CpG sites at promoter region of human WNT-2 gene in HTR-8/SVneo. (Each circle shows the average methylation of an individual CpG site; open circle=0% methylation, the last circle of average =percent of total methylation per sample). (E) Total percent of methylation levels (%) of 23CpG sites of human WNT-2 gene. N=3. (F) BPA reduced protein expressions of WNT-2 and β-catenin in HTR-8/SVneo. (G) (H) Gray values were qualified. N=3. (I) 5-aza-dc treatment altered protein expressions of DNMTs, WNT-2, and β-catenin in HTR-8/SVneo cells. (J) (K) Gray values were qualified. N=3. (L) 5-aza-dc treatment reduced DNA methylation of 23CpG sites at promoter region of human WNT-2 gene in HTR/SVneo-8 cells. (Each circle shows the average methylation of an individual CpG site (open circle=0% methylation, the last circle of average =percent of total methylation per sample). (M) Total percent of methylation levels (%) of 23CpG sites of human WNT-2 gene was analyzed. N=3. (N) BPA exposure attenuated trophoblasts-endothelial cell interaction: HTR-8/SVneo cells (red) and HUEVC cells (green). (O) Quantification of trophoblasts-endothelial cell interaction by measuring the red and the green fluorescence portion. N=3.

Quantitative data are presented as mean ± SEM. Students t test was performed to determine the statistical significance of two comparisons. *P<0.05, **P<0.01, ***P<0.001.

In HTR-8/SVneo cells, BPA evidently up-regulated DNMT-1 expression (Fig3.B; C) at levels higher or equal to 1μmol/L, while expressions of DNMT-3a and DNMT-3b were not affected (Fig3.B). The average DNA methylation level of WNT-2 was elevated at BPA levels higher or equal to 0.1μmol/L (76.70% ± 1.5 vs 44.5% ± 1.4, P<0.01) (Fig3.D; E) and the affected CpG sites were at CpG1, CpG2, CpG8, CpG9, CpG10, CpG11 and CpG14 (Fig3.D). Meanwhile, the protein expressions of WNT-2 and β-catenin were down-regulated at BPA levels higher or equal to 10μmol/L (Fig3.F; G; H). Overall, BPA exposure increased DNA methylation and reduced expression of WNT-2 and β-catenin in HTR-8/SVneo cell, in accordance with results in mice.

To establish a direct role of DNMT-1 in WNT-2 and β-catenin expression, we treated HTR-8/SVneo cells with DNA methylation transferases inhibitor, 5-aza-dc. Inhibition of DNA methylation transferases effectively reduced DNA methylation of WNT-2 (5aza-dc vs controls: 83.9% vs 66.5%; P<0.01) (Fig3.L; M) and the affected CpG sites were at CpG1, CpG2, CpG4, CpG9, CpG10 and CpG14 (Fig3.L). Meanwhile, the protein expressions of WNT-2 and β-catenin were upregulated (Fig3. I; J; K).

BPA impeded interaction between HTR-8/SVneo and HUVEC

BPA exposure impaired interaction between HTR-8/SVneo and HUVEC cells at levels higher or equal to 1μmol/L revealed by a lower ratio of HTR-8/SVneo/HUVEC in the tube-like structures (1μmol/L BPA vs control: 35.38%±1.82% vs 55.93%±2.57%, P<0.01) (Fig3. N; O), in accordance with results in mice.

Discussion

To our knowledge, this is the first study to demonstrate preeclampsia-like features following prenatal oral exposure of BPA in a mouse model. The development of preeclampsia occurs in two stages: it is first initiated by reduced placental perfusion resulting from insufficient spiral artery remodeling, then impaired placentation causes systemic pathophysiological changes in the maternal circulation. This study showed changes in maternal circulation including elevated SBP, increased Flt-1/PlGF ratio and kidney damage, although urinary protein level was not affected in mice exposed to BPA. BPA exposure reduced invasion of trophoblasts in vessels of placental section in mice. We understand that mouse is not an ideal model to study placental vascular remodeling as the trophoblast cells only invade the maternal side of the junctional zone. Therefore, we further investigated the trophoblasts invasion upon BPA exposure by using human placental line. We found that BPA exposure inhibited ability of HTR-8/SVneo to invade and replace endothelium, indicating that BPA exposure in humans may lead to poor uterine artery modification by trophoblasts. Moreover, we demonstrated that BPA exposure in vitro in a human cytotrophoblast cell line negatively impacted cytotrophoblast invasion and highlighted the possible molecular mechanism that BPA exposure down-regulated WNT-2 by inducing hypermethylation of WNT-2 via DNMT-1.

Physiological cytotrophoblast invasion must be tightly regulated to ensure that the depth of cytotrophoblast invasion proceeds to the appropriate extent but no further. Shallow invasion and insufficient vessel remodeling are characteristics of preeclampsia. Impaired trophoblast invasion and placental vessel remodeling were showed in our model of BPA exposure induced preeclampsia like model. Immunofluorescence staining of cytotrophoblast and smooth muscle cells in mice placentas revealed that BPA exposure inhibited the trophoblasts invasion, leading to vascular smooth muscle retention and consequently reduced decidual vascular areas, consistent with previous findings of BPA exposure impairing placental labyrinth and spongiotrophoblast layers in mice^{3, 11}. BPA did not alter proliferation of placental cells in the BPA-induced preeclampsia model in our study. In contrast, at much higher exposure levels (50 mg/kg/d) than 4 μM (relevant to182.5 μg/kg/day) used in this study, BPA exposure induced placental cell degeneration and necrosis in mice³, implying that high concentration of BPA has obvious toxic effects on placental cells.

There is accumulating evidence about the role of BPA in defecting trophoblast biological processes in vitro. Spagnoletti et al. found BPA inhibited HTR-8/SVneo migration and invasion but did not affect trophoblast/HUVEC interaction at a level of 10⁻⁵μmol/L after 48h exposure¹², while we found BPA impaired trophoblast/HUVEC interaction at a level of 1μmol/L after 14 days exposure in the present study, indicating that either chronic or higher dose exposure might be essential for this effect of BPA. Moreover, another study found that 24h BPA exposure reduced a trophoblast cell line, BeWo, cell viability at a dose of 10 μmol/L¹³, while we did not observe reduced HTR-8/SVneo cell viability until 100 μmol/L BPA treatment. In primary human trophoblast cells, 24h BPA exposure increased cell apoptosis and necrosis at a dose of 0.02 μg/ml¹⁴. These results demonstrated a different level of susceptibility to BPA exposure in these cells. We speculate that it might be due to the different level of syncytialization among these cells. Interestingly, the results in a recent study consistently showed BPA exposure (<50 μmol/L) did not change the cell viability in HTR-8/SVneo cells at similar doses used in the present study¹¹. However, their finding of enhanced cell invasion and migration by BPA exposure is inconsistent with Spagnoletti’s findings¹². The authors suspected that it might be due to different culture conditions. Altogether these evidences, including our results, support the adverse effects of BPA on trophoblast invasion, the underlying biological mechanism of BPA exposure induced preeclampsia-like features in mice.

Although current findings are inconsistent, our conclusion above was supported by both systematic and molecular examinations in our model. Systematic evidence is the observation of preeclampsia-like features and relevant placental pathophysiological changes. In this study, pregnant mice treated with BPA developed comprehensive diagnostic criteria for preeclampsia, including elevated SBP, increased ration of Flt-1/PlGF and impaired kidney glomerular though proteinuria remained unaffected. Considering proteinuria is not essential for the diagnosis of preeclampsia¹⁵, our diagnosis is reliable. Although proteinuria is a common sign and risk factor of kidney impairment, the number of albuminuria does not completely reflect the severity of renal disease. For example, when the proteinuria is less frequent, it is possible that the renal lesion is improved or worsen due to glomerular fibrosis and reduced filtration protein, which was observed in the current study.

Non-pregnant female mice treated with BPA did not develop preeclampsia-like features in our study. In contrast, Saura, M reported elevated blood pressure in non-pregnant mice treated with 0.4 μmol/L BPA for 30 days¹⁶. We speculate that the difference between our findings might be due to the exposure duration, implying that short-term BPA exposure in a non-pregnant state is insufficient to cause endothelium dysfunction and vascular constriction, leading to hypertension, and that short-term BPA exposure during pregnancy causes elevated blood pressure probably due to the presence of placenta. These data demonstrate pregnancy-specific effects of BPA in inducing preeclampsia-like features in mice, strongly suggesting it occurs via placental alteration. Indeed, we observed imbalanced placental angiogenesis and anti-angiogenesis factors in BPA exposed pregnant mice, manifested by elevated ratio of circulating and placental Flt-1/PlGF, and impaired placental vessel remodeling. Additionally, BPA exposure is associated with many other pregnancy complications associated with abnormal placentation, including spontaneous preterm birth¹⁷, small for gestational age¹⁸ and pregnancy loss¹⁹, highlighting the role of BPA in abnormal placentation. Further studies are warranted to evaluate the impacts of BPA on other placental disorders, including preterm birth, recurrent pregnancy loss, and fetal outcomes such as IUGR, even though we did not observe preterm birth and IUGR in this present study.

The molecular evidence is the observation of dysregulated signaling pathways critical for trophoblast invasion by BPA exposure. WNTs are a family of secreted glycoproteins with diverse roles in development and plays vital roles in human trophoblast invasion^{20, 21}, partly by inducing MMP-2 and MMP-9 expression²². Reduced expression of WNT-2 impaired trophoblast invasion and relates to increased risk of preeclampsia²³. In our BPA-induce preeclampsia-like mouse model, we found reduced expression of WNT-2, β-catenin, MMP-2, MMP-9, and elevated gene expression of TIMP-1 and TIMP-2 in placentas, strongly suggesting that BPA inhibits trophoblast invasion through canonical WNT signaling. Expression of WNT-2 can be down-regulated by reprogramming of DNA methylation in human placenta²⁴. In our study, we found a negative association between DNA methylation and expression of WNT-2 in mice placenta and HTR-8/SVneo cell upon BPA exposure. Epigenetic modifications include DNA methylation, histone modification and phosphorylation, which are regulated by corresponding epigenetic modification enzymes. Through PCR-array analysis of epigenetic modification enzymes, we identified that DNMT-1 was the most affected enzyme in BPA-induced preeclampsia-like mouse model while DNMT-3a, DNMT-3b, TET-1, TET-2 and TET-3 were not affected, indicating the role of methylation reprogram mediated by DNMT-1, which is over-expressed in the placenta of preeclampsia women²⁵. Using HTR8/SVneo cells, we confirmed that BPA exposure down-regulated WNT signaling through increased DNA methylation in promoter region of WNT-2 gene mediated by DNMT-1. In a previous study, BPA also inhibits WNT/β-catenin pathway in neural stem cells²⁶. However, BPA was reported to activate β-catenin in trophoblast of labyrinthine and spongiotrophoblast in mice placenta at exposure level of 0.5 mg/kg/d from E1 to E11²⁷. One possible explanation for this difference from our findings may be the different exposure and observation window.

The strength of this study is the comprehensive evaluation of our observations. However, there are some shortages in our study. Firstly, total BPA intake could not be assessed as daily water intake was not measured, thus we could not calculate the exact BPA exposure level. In fact, 24h urinary BPA concentrations were measured to evaluate exposure level and it was found that urinary BPA levels rose progressively with increasing does of BPA, confirming that our methods were sufficient to distinguish and determine BPA exposure levels. Secondly, the other weakness of our study is the lack of long-term effects of BPA exposure on pregnancy outcomes in our mouse model, which warrants future research.

Conclusions

We demonstrated that BPA exposure during pregnancy impedes trophoblast invasion and placental spiral artery remodeling, leading to preeclampsia-like features in mice. BPA inhibited trophoblast invasion and placental vessel remodeling through mechanisms involving in down-regulation of MMPs and up-regulation of TIMPs, and down-regulation of WNT signaling through increased DNA methylation in the promoter region of WNT-2 gene mediated by DNMT-1. Our study provided new evidence for a causal relationship between BPA exposure and preeclampsia. Therefore, our study suggests that reducing or avoiding BPA exposure during pregnancy may reduce the risk of preeclampsia and improve maternal and child outcomes.

Perspectives

We identified that 4 μmol/L was the minimum effective concentration to induce development of preeclampsia-like features in mice, which corresponds to a daily intake of about 182.5 μg/kg bw/day considering a mean body weight of 20 g and a daily intake of about 4 milliliter water in adult mice²⁸. This value is three times higher than the U.S. Environmental Protection Agency’s (EPA) recommended safe daily BPA exposure (50 μg/kg bw/d)²⁹, but much lower than the current FDA recommended lowest-observable-adverse-effect level (NOAEL) of 5 mg/kg bw/d³⁰. Though the estimates of daily average BPA intake in human adults are 0.4–1.4 μg/kg.bw/d³¹, bad inhabits, such as consumption of canned soup daily lead to >1000% increase of BPA intake³². Thus, BPA exposure of 4 μmol/L should occur and have potential adverse effects on human. Accounting that the major exposure way of BPA is through diet, changing behaviors, such as using BPA-free products, could potentially reduce BPA exposure and risk of preeclampsia. Understanding the role of BPA exposure in preeclampsia and placentation not only provide values of disease prevention but also help to answer the questions about the reproductive toxicities of BPA.

Novelty and Significance:

1). What Is New

This is the first study to demonstrate BPA exposure during pregnancy affects placental development and lead to preeclampsia-like features in a mouse model.

2). What Is Relevant

These data shows that BPA exposure inhibits WNT signaling to impede trophoblast invasion and placental spiral artery, leading to preeclampsia-like features in mice.

3). Summary

This study provides values that BPA is a placenta toxicant. Behavior interventions to reduce

BPA exposure during pregnancy could potentially help to reduce risk of preeclampsia.