The impacts of intrauterine Bisphenol A exposure on pregnancy and expression of miRNAs related to heart development and diseases in animal model

This study aimed to examine the impact of BPA exposure on pregnancy and foetuses on cardiac tissues and the expression of cardiac microRNAs (miRNAs) related to heart development and diseases. Pregnancy is known to be the “critical windows” in determining the offspring physical and cells development in their life after birth. The increment of the risk of cardiovascular disease (CVD) in a later stage of life has been reported by few studies demonstrated from prenatal exposure of BPA. BPA has been shown to alter miRNAs expression profiles for organ development, regeneration and metabolic functions. These alterations have been associated with the risk of CVDs. However, the associations between pregnancy outcomes and miRNAs expression in cardiac of mother- and foetuses-exposed to BPA are still not entirely explored. In BPA-exposed pregnant rat groups, a significant weight gained was observed in comparison to control (p < 0.05). Interestingly, significant changes in systolic and diastolic blood pressure between the first and third trimester of BPA-exposed pregnant rats were also observed (p < 0.05). In BPA-exposed pregnant rats, miR-499-5p was significantly altered in the heart (p < 0.01). Meanwhile, altered miR-17-5p, -208-3p, and -210-3p expressions were observed in all heart of the foetuses from BPA-exposed pregnant rats (p < 0.05). In H&E staining, BPA-exposed foetal hearts showed a sign of fibrosis while BPA-exposed pregnant rats showed muscle remnant. Masson trichrome staining further confirmed the presence of fibrosis observed in BPA-exposed foetal heart and reduced expression of cardiac troponin I (cTnI) was also observed in BPA-exposed foetal heart. In summary, altered cardiac miRNAs with histological changes were observed in both mother- and foetus-exposed BPA These findings put forward the importance of future work to further understand how prenatal BPA exposure affect foetuses in their later stage of life.


Main Text
Materials and methods. Chemicals. Tween-80 and BPA were purchased from Sigma Aldrich. In this study, Tween-80 was used as vehicle control for treatment (with 0.4% of Tween-80 in total solution). BPA was dissolved in Tween-80 (with the same volume) and prepared to 20 ppm concentrations. For liver histology standard protocol, paraformaldehyde (PFA) and phosphate buffer solution (PBS) were used and were purchased from Sigma Aldrich and Thermo Scientific, respectively.
Animals experimentation. Adult female rats aged six to eight weeks (Sprague Dawley) (weighing between 200-250 g, n = 5) were housed individually and allowed free access for food and drinks. Females were mated with males rat in ratio 1:1 in different cages. The vagina smears were collected the next morning to confirm positive mating 34 . The pregnant rats were grouped into three categories namely drinking water containing vehicles (0.4% Tween-80 35 ), water containing 5 ppm BPA and 20 ppm BPA (0.05 mg/ml and 0.2 mg/ml, respectively). Glass water bottles were used to avoid potential contamination from sources other than administration. The water intake was noted with an estimation of consumption approximately 25 mL or more per day. The treatment began on pregnancy day 2 (PD2) until pregnancy day 21 (PD21). The treatment began at PD2 to observe any significant effect on organogenesis 36 . This study was conducted in accordance with relevant guidelines and regulations and is approved by Universiti Teknologi MARA (UiTM) Committee of Animal Research and Ethics (Approval Number UiTM CARE: 222/7/2017 (8/12/2017)).
Throughout the treatment period, the weight of the pregnant rats was taken four times (on PD2, PD7, PD14 and PD21) whereas blood pressure (BP) was recorded twice during early (PD2) and end (PD18) of the pregnancy. BP readings were taken twice to minimise stress factor in the treated rats as it may contribute to number of factors such as miscarriage. Apart from that, lighting and temperature of the animal room were also monitored and controlled. Food intake was measured four times throughout the treatment period in which each pregnant rat was supplied with approximately 230 g of normal diet pellet.
Non-invasive BP measurement on the pregnant rats were conducted using volume pressure recording (VPR) sensor technology according to the methodology described by Wang et al. 37 . The rats were placed in a strainer with the nose protruded through the front of the nose cone to allow comfortable breathing while the tail was fully extended and exited through the rear hatch opening of the holder. The blood flow and blood volume in the tail were measured by VPR system and generated BP readings simultaneously.
Caesarean section was performed on PD21 according to the previous protocol 38 . Each litter of rats were weighed, and the size was measured. The number of foetuses for each rat was recorded (n = 8-12 pups/rat). Both pregnant and foetuses heart were isolated, placed in RNAlater buffer (QIAGEN, Dusseldorf, Germany) and stored in 4 °C. The samples were then stored at −80 °C on the following day. The livers were collected from both the mothers and the foetuses for histology and examination (H&E).
www.nature.com/scientificreports www.nature.com/scientificreports/ Liver histology and examination. Lobes of the mothers and foetal liver were fixed in 4% PFA in 0.1 M PBS, dehydrated, and embedded in paraffin. The livers were sectioned with 5 µm sections and transferred to microscope slides (Fisher Scientific, Whitby, ON). Sections were stained using the standard H&E protocol for histological analyses as described in a previous study 39 .
Heart histology and examination. Mothers heart were cut longitudinally while the total heart of the foetuses was used and fixed in formalin, dehydrated and embedded in paraffin. The hearts were sectioned with 5 µm sections and transferred to microscope slides (Fisher Scientific, Whitby, ON). Sections were stained using H&E protocol for histological analyses as described in a previous study 39 .
Sections were also stained for Masson Trichrome (MT) and immunofluorescence staining. For Masson Trichrome, the 5 µm sections of fetal hearts were deparaffined and stained according to the Masson Trichrome kit (Bio-Optica, Milano) prior to view.
Immunofluorescence staining was done by blocking the sections of fetal heart for 30 min with 1% horse serum in PBS, then incubated at room temperature for 3 hrs with primary antibody of troponin (1:400). Sections were washed thrice with PBS for 5 min and secondary antibody incubation was carried out using 1:500 fluorescein isothiocyanate (FITC). Subsequently they were washed thrice with PBS in 5 min. Tissues were counterstain with DAPI for 1 min, washed as previous. Tissues then were mounted with prolong gold antifade reagent and cover slipped prior to view under fluorescence microscope (Olympus, New York) and confocal microscope (Leica, UK) 40 . miRNA extraction. In brief, the isolated samples were removed from RNAlater buffer (QIAGEN, Dusseldorf, Germany) and homogenised using a mortar and pestle 41 . miRNA was extracted from the heart samples using miRNeasy mini kit (QIAGEN, Dusseldorf, Germany). Approximately, three to four foetal hearts from each group or 40 mg of homogenised tissue was used for extraction. Extracted RNA was quantified using a Nanodrop spectrophotometer and aliquoted into single-use aliquots and stored at −80 °C. Total RNA was extracted from ∼40 mg of heart tissue using the AllPrep miRNA Universal Kit following the manufacturer's instructions. miRNA polymerase chain reaction array (miRNA-PCR). Total RNA of 250 ng from tissue was used for cDNA synthesis using miScript RT II cDNA Synthesis Kit (QIAGEN, Dusseldorf, Germany) as per manufacturer's protocols. miRNAs abundance was measured by miRNA PCR array using Applied Biosystems Fast Advanced Master Mix (QIAGEN, Dusseldorf, Germany). PCR was performed and the target genes were normalised to three chosen reference genes. SNORD68, SNORD96A and RNU6-6P were employed as the reference genes to analyse miRNAs expression in the rat cardiac tissues [42][43][44] . The ratio between the target gene and the geometric mean of the reference genes were calculated to achieve mean normalised expression (MNE) of the target genes in each sample. For analyses, Ct values > 35 cycles were excluded from the analyses if duplicates had a standard deviation greater than 1 cycle. Final probe concentrations were maintained as suggested by the manufacturer in a 20 μl reaction. Sample concentrations were 1:10 dilution of the stock pre-amplification generated product as per the manufacturer's protocol. Covariation (CV) of the reference genes was determined from Biorad CFX Manager 3.1 that generate the stability values. CV value is an indicator of variation in a group, the less CV value showed low variation among the groups. From the data, the CV values of both reference genes of rat and foetus cardiac tissues were below 0.5, which indicates the stability of these genes is accurate and reasonable. miRNA PCR data was analysed using 2-ΔΔCT method.
The list of targeted miRNAs and their ID number were as tabulated in Table 1.
Statistical analysis. Non-parametric analysis was carried out using SPSS version 20 (SPSS, Inc.). Kruskal Wallis test was used where appropriate to compare control against BPA-exposed values with alpha set at p ≤ 0.05. Data are shown as mean ± SEM in miRNA expression while other data are shown as mean ± SD.
Results. BPA has no effect on the mothers and foetuses liver. Standard histology of the liver was performed to determine the effect of prenatal BPA exposure to structural liver maturation of foetus from PD2 to PD18. Upon miRNA ID number NCBI Accession ID Role of miRNA www.nature.com/scientificreports www.nature.com/scientificreports/ observation, we found that there were no visible differences in the structure between control and BPA-exposed in both pregnant and foetal liver (Fig. 1).
The H&E-stained liver sections of the control and BPA-exposed rats revealed normal liver histology, displaying a normal cell size, with a prominent cell nucleus, uniform cytoplasm, and radially aligned sinusoid and central vein. The similar structure pattern in the control and BPA-exposed foetus liver indicated that prenatal BPA exposure from PD2 to PD18 has no toxicity effect on the liver.
Exposure of BPA affects the histology of BPA-exposed pregnant rats and foetal heart. Histology of the mother's and foetus heart was performed to compare the morphology of the muscle cells in control and BPA-exposed subjects (Fig. 2). The H&E heart sections showed a sign of fibrosis in the BPA-exposed foetus heart. Meanwhile, in the mothers, the heart of the BPA-exposed pregnant rats showed muscle remnants.
BPA exposure during pregnancy affects the mothers' weight gained and blood pressure. In the pregnant rat, systolic blood pressures (SBP) and diastolic blood pressure (DBP) were measured at PD2 and PD18 (Fig. 3A). The difference in the SBP and DBP were found to be significant within the BPA-exposed groups (p < 0.05). The results indicated that both SBP and DBP significantly increased during pregnancy in both groups of BPA-exposed rats as compared to the control rats (p < 0.05). When the BPA-exposed groups are compared with the control group, the SBP (in the third trimester) was raised up from 125 ± 9.505 mmHg in control rats to 151 ± 4.50 mmHg and 137 ± 10.46 mmHg in the 20 and 5 ppm BPA-exposed rats, respectively (p = 0.009). DBP progressively increased significantly during third trimester in the BPA-exposed groups from 92 ± 9.86 mmHg in the control group to 113.00 ± 2.54 mmHg and 105.00 ± 9.35 mmHg in the 20 and 5 ppm of BPA, respectively (p = 0.028, p = 0.007).
The differences between BP reading from PD2 and PD18 is shown in Fig. 3B. The results indicated that SBP increased significantly in the 20 ppm (56.4 ± 9.902 mmHg) BPA-exposed pregnant rats in comparison to control (10.0 ± 10.775 mmHg) pregnant rats. Meanwhile, DBP also showed the similar pattern in SBP increment of 20 ppm (21.0 ± 2.534 mmHg) BPA-exposed pregnant rats in comparison to control (17.0 ± 6.979 mmHg) pregnant rats.
All mothers significantly gained weight from PD2 to PD18 regardless of whether they were given vehicle control or being exposed to BPA (Fig. 4A). This suggested that all mothers are healthy throughout the treatment. As compared to the control, weight gained of BPA-exposed rats with 5 ppm (54 ± 19.61 g) was found to be significant (p = 0.025) and 20 ppm (135 ± 39.95 g) was nearly significant (p = 0.053). Interestingly, the significant difference in weight gained was observed between both BPA-exposed groups (p = 0.001). Apart from that, no significant differences were observed for the drinking pattern in either control or BPA-exposed rats (Fig. 4B). Although changes in the foetuses weight and size were observed, however, there were no significant differences in both parameters between control and BPA-exposed foetuses (Fig. 5A,B, respectively). BPA shows no effect on mothers and foetuses liver histology. Representative of liver section of control (A) and BPA-exposed (B) pregnant rat, Foetal liver section at PD21 from pregnant rats of control (C) and BPAexposed (D) stained with hematoxylin and eosin dye (10X magnification). (2020) 10:5882 | https://doi.org/10.1038/s41598-020-62420-1 www.nature.com/scientificreports www.nature.com/scientificreports/ Bisphenol A prenatal exposure alter foetuses cardiac miRNAs involved in development and diseases. miR-17-5p, -21-5p and -133a-3p have been reported to be upregulated during the incidence of cardiac disease. These miR-NAs are important in cardiomyocytes proliferation and development 45 . In this study, significant elevation of miR-17-5p expression was observed in 5 ppm (1.50 ± 1.14) and 20 ppm (2.25 ± 1.34) compared to the control (p = 0.027) (Fig. 6).
Cardiac fibrosis and expression of cardiac troponin I in BPA-exposed foetal heart. In here, non-prominent presence of fibrosis was observed in heart of pregnant rats of BPA-treated groups compared to control (Fig. 9). As for foetus, Masson trichrome staining further confirmed the appearance of fibrosis observed in H&E staining (Fig. 10), BPA-exposed foetal heart showed signs of fibrosis (in red circle) based on the observation. However, more prominent sign of fibrosis was confirmed in 20 ppm of BPA-exposed foetal heart compared to 5 ppm and control. The expression of cardiac troponin I (cTnI) was reduced in BPA-exposed foetal heart compared to the control of foetal heart (Fig. 11).

Discussion. Many in vivo and in vitro studies have investigated the detrimental effects of BPA exposure in
the early development of different organs and cell types. Recently, BPA has been shown to affect the normal function of in vitro fetus heart model 46 . In the liver, abnormalities in its function and damage were reported in rat offspring of BPA-exposed pregnant rats 47 . The study demonstrated that the liver abnormalities and damage were only observed at the postnatal of week 21 offspring from BPA-exposed pregnant rats. Meanwhile, in our study, we showed that prenatal exposure of 5 ppm and 20 ppm BPA had no significant effects on the liver morphology of the foetuses and pregnant rats. The structure of the liver portrays normal hepatic function suggesting that prenatal BPA exposure at these doses does not exert hepatotoxicity. Although the doses used in this study are high (5 ppm and 20 ppm), these high BPA concentrations have been suggested as estimated daily intake based on data extrapolated from urinary BPA levels 48 . A previous study on reproductive organs observed no increment of cancer in 0.015-75 ppm and 750-7500 ppm of BPA exposure 49 . However, other studies documented that BPA foetal exposure at the range of environmental (less than 1 ppm) and high concentration (15 to 150ppm) showed a correlation of BPA concentration with its detrimental effects on the embryos 50,51 . Similarly, this study demonstrated the effect  www.nature.com/scientificreports www.nature.com/scientificreports/ of BPA in both the mothers and foetus hearts, whereby histology on the heart of the BPA-exposed pregnant rats showed injury in the muscle which could be due to the necrotic process or cardiomegaly. Meanwhile, in the foetuses, histology examination showed a sign of fibrosis compared to the control. These suggested that BPA might give an impact on foetal development within the chosen concentrations.
Nevertheless, this study revealed a correlation between positive weight gain in the BPA-exposed groups with raised SBP readings throughout the treatment (Fig. 3). Increased body weight has been identified as a risk factor for hypertension development during pregnancy 52 . This could be due to the rise in cardiac output and heart rate. Recently, researchers have reported the association of CVD with hypertension in the BPA-exposed individual. These two chronic diseases are very common as one of the public health problems 53 . Thus, Han et. al (2016) Figure 6. miR-17-5p was upregulated in foetuses heart of BPA-exposed pregnant rats. miRNA expression of (A) miR-21-5p (B) miR-15b-3p (C) miR-133a-3p (D) miR-17-5p were measured in the heart of foetuses of control and BPA-exposed pregnant rats. All the miRNA have been reported involved in cardiac development. Data are expressed as means. (n = 5 rats per group). *p < 0.05; **p < 0.01; ***p < 0.001. Figure 7. miR-208a-3p and miR-210-3p were upregulated in foetuses heart of BPA-exposed pregnant rat. (A) miR-208a-3p (B) miR-30a-5p (C) miR-499-5p (D) miR-210-3p expression were measured in foetuses heart of control and BPA-exposed pregnant rats. Altered expression in these miRNAs have been associated with increase risk of cardiac disease development. Data are expressed as means. (n = 5 rats per group). *p < 0.05; **p < 0.01; ***p < 0.001.

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(2020) 10:5882 | https://doi.org/10.1038/s41598-020-62420-1 www.nature.com/scientificreports www.nature.com/scientificreports/ conducted a study to investigate the association between BPA exposure and CVD development of in a randomized clinical trial. The study observed an increment in SBP with 4.5 mmHg after the exposure to BPA. Our results were in agreement with those findings where we showed changes in BP in BPA-exposed pregnant rats compared to the control pregnant rats. Although the mechanisms of associations between BPA, hypertension and CVD were unclear, it could occur due to several factors such as endocrine disruptor, oxidative stress and inflammation induction, links with other chronic diseases and epigenetic changes.
As shown in Fig. 4A, all rats gained weight throughout the treatment period. However, a significant weight gain was observed after the exposure of 5 ppm BPA compared to the control pregnant rats (p = 0.025). This observation was in line with few reported studies, which demonstrated an increment of weight in those exposed with BPA 8,54,55 . Exposure to an endocrine-disrupting chemical such as BPA promotes adipogenesis and weight gain. This made BPA as a potential determinant of obesity 56 . This study also confirms the obesogenic impacts on male mice after BPA exposure. In contrast, another study reported no significant differences in weight between vehicle control and BPA groups 57 . The study was conducted on Sprague-Dawley rats from gestational day 6 until postnatal day 90. They observed no significant increment on the gestational length and bodyweight of the pups exposed to BPA. This is in line with the results reported in this study (Fig. 5B). They interpreted that certain BPA dosage might not affect the endpoint results although few previous studies reported on the effect of BPA exposure on weight gain.   www.nature.com/scientificreports www.nature.com/scientificreports/ Epigenetic modifications are known to be related to diseases such as CVD and there are many studies have already linking epigenetic changes with BPA exposure 58 . Based on these findings, we further investigated the impact of BPA exposure on miRNAs. Three out of eight targeted cardiac-related miRNAs exhibited altered expression in foetuses of BPA-exposed group. This observation is in agreement with a study that was conducted by Barry et. al. (2013) in investigating the toxicity levels of BPA in rats 57 . Previous study observed overexpression of miR-21 in BPA-induced 3T3-L1 cells as it has been reported to be an important gene in the regulation of insulin and glucose 59 . Nevertheless, previous study also reviewed the abnormally expressed miR-21-5p in cancer development such as breast, lungs and colorectal 60 .
miR-133a-3p and -208a-3p are involved in cardiomyocytes proliferation and differentiation 45 . As we were focusing on the possibility of the offspring to develop cardiac disease, miRNAs related to cardiac disease were chosen. Interestingly, miR-208a-3p was elevated in foetuses with 20 ppm BPA-exposed pregnant rat (p = 0.039). miR-208a-3p is involved in cardiac contractility and function, where the expression of this gene in response to modulation of myosin heavy chain results in the upregulation of βMHC. miR-208a-3p is a cardiac-specific  www.nature.com/scientificreports www.nature.com/scientificreports/ miRNA and has been reported to be an important miRNA in cardiomyocyte hypertrophy and fibrosis 3 . This gene is absent in healthy subject thus, the elevation of its expression suggested a modification in the heart of the foetus exposed to BPA.
This study also focuses on the impact of BPA exposure on the heart of mothers and foetuses, by H&E, Masson trichrome (MT) staining, cTnI expression and cardiac miRNA profile. Our findings showed that elevated expression of miR-208-3p (in foetal heart) and miR-499-5p (in mothers heart) after exposure to BPA could be associated with the expression of cardiac troponin I (cTnI) (Fig. 11). This findings are in agreement with previous reported studies which associated the upregulation of miR-208a and miR-499 with cTnI expression 7,61,62 .
In agreement with our findings, miR-17-5p is elevated in foetus-exposed to BPA, thus, suggested the possibility of the offspring to develop heart disease later in life. There was study documented on a role of miR-17 in liver and cardiac fibrosis 63 . miR-210-3p is significantly expressed in the BPA-exposed foetus heart, which suggests that Figure 11. BPA exposure effects foetal cardiac troponin I (cTnI) expression. Immunofluorescence staining of foetal heart section subjected to cardiac troponin I expression. (A) control foetal heart, (B) 0.05 mg/ml BPAexposed, (C) 0.2 mg/ml BPA-exposed foetal heart, stained with DAPI and FITC. ZF 2.6 (n = 8). Scale bar 25 µm and 75 µm.

Scientific RepoRtS |
(2020) 10:5882 | https://doi.org/10.1038/s41598-020-62420-1 www.nature.com/scientificreports www.nature.com/scientificreports/ this miRNA has an impact on angiogenesis and fibrosis 64 . In parallel, a study reported similarities between foetal and failing heart in which elevated miR-210-3p was observed, indicating the prevalence of heart failure development 65 . In our findings, this gene is significantly altered in 20 ppm of BPA-exposed foetus heart. Moreover, our H&E and MT staining indicated fibrosis in both BPA exposed groups compared to control group. However, the fibrosis was more prominent in 20 ppm BPA. Previously, BPA shown to upregulate the expression of marker for fibrosis such as interleukin-6 (IL-6) and collagen type 1 66,67 . In here, our result is the first to suggest that BPA exposure during prenatal life may altered cardiac miRNAs and induced fibrosis.
conclusions. There is increasing evidence that exposure to BPA during the intrauterine environment may play a significant role in the global health problems specifically in the development of CVD. BPA has the potential to induce obesity and hypertension in the exposed individual during pregnancy. As the foetus is partially protected from the adverse effect of environmental conditions, in utero exposures of BPA may give impact to the foetus indirectly. Furthermore, our data suggest that altered miRNA expression in the foetus do affect the heart tissues. Thus further study need to be done on understanding whether these changes seen in BPA-exposed foetus could increase the risk of CVD in offspring later in life.

Limitation
There are some limitations to this study. Firstly, this study focused on the impact of miRNAs expression in the foetal hearts only and does not extend until in postnatal life. However, the expression during postnatal life could be influenced by other factors such as environmental and nutrient intake that could interfere with the overall findings. Secondly, the number of the sample size used in this study is limited to 5. The expression could be more significant if the sample size is bigger than 5.

Data availability
The datasets used and/or analysed in this study are available from the corresponding author on reasonable request.