Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Sep 1.
Published in final edited form as: Reprod Toxicol. 2023 Jul 22;120:108446. doi: 10.1016/j.reprotox.2023.108446

Exposure to di-isononyl phthalate during early pregnancy disrupts decidual angiogenesis and placental development in mice

Arpita Bhurke 1,3, Juanmahel Davila 1, Jodi A Flaws 1,3, Milan K Bagchi 2,3, Indrani C Bagchi 1,3,*
PMCID: PMC10683654  NIHMSID: NIHMS1923019  PMID: 37482143

Abstract

Di-isononyl phthalate (DiNP), an endocrine-disrupting chemical, is found in numerous consumer products and human exposure to this phthalate is becoming inevitable. The impact of DiNP exposure on the establishment and maintenance of pregnancy remains largely unknown. Thus, we conducted studies in which pregnant mice were exposed to an environmentally relevant dose (20 μg/kg BW/day) of DiNP on days 1 to 7 of gestation, then analyzed the effects of this exposure on pregnancy outcome. Our studies revealed that exposure to DiNP during this window led to fetal loss towards the end of gestation. Further studies showed that, although embryos were able to attach to the uterus, implantation sites in DiNP-exposed uteri exhibited impaired differentiation of stromal cells to decidual cells and an underdeveloped angiogenic network in the decidual bed. We also found that exposure to this phthalate has a significant effect on trophoblast differentiation and causes disorganization of the placental layers. The labyrinth was significantly reduced, resulting in compromised expression of nutrient transporters in the placentas of mice exposed to DiNP. These placental defects in DiNP-exposed females were the cause of fetal loss during the later stages of gestation.

1. INTRODUCTION

Di-isononyl phthalate (DiNP) is an endocrine-disrupting chemical commonly found in plastic products such as storage containers, medical devices, packaging, hoses, lubricants, fabrics, and toys [1, 2]. Since phthalates are not covalently bound to the plastic polymers, they can leach out of these products. Humans can be exposed to phthalates via ingestion, inhalation, and dermal contact [3–5]. Metabolites of DiNP have been detected in blood, urine, semen, amniotic fluids, and breast milk [3–5]. While the literature on daily exposure to DiNP is sparse, occupational exposure to DiNP may be as high as 26 μg/kg body weight (BW)/day [6]. Epidemiological studies in women show that DiNP exposure results in adverse pregnancy outcomes [5, 7, 8]. It has been reported that women with high serum DiNP levels took a long time to become pregnant [8, 9]. Additionally, an increased incidence of miscarriage has been reported in women occupationally exposed to high doses of phthalates [5].

Studies using rodent models provide insights into the physiological processes by which phthalates interfere with reproductive functions. A previous study investigating the impact of DiNP (20, 100, 20 mg/kg/day, 20, or 200 μg/kg/day) on female reproduction, observed that about 33% of the females exposed to 20 μg/kg BW/day dose of DiNP were unable to maintain pregnancy [10, 11]. While the literature on daily exposure to DiNP is sparse, occupational exposure to DiNP may be as high as 26 μg/kg body weight (BW)/day, making 20 μg/kg BW/day dose of DiNP an environmentally relevant dose [6]. While most studies suggest that the reproductive system is a target of phthalate toxicity, the effect of phthalate exposure on uterine function during pregnancy remains largely unknown. Therefore, the aim of this study is to specifically examine the impact of exposure to a dose (20 μg/kg BW/day) of DiNP that is comparable to human exposure levels.

Epidemiological studies have observed a correlation between urinary levels of DiNP and adverse pregnancy outcome [5, 7, 8]. It has been reported that women with high serum DiNP levels took a long time to become pregnant [8, 9]. To our knowledge, this is a first study investigating the impact of exposure to 20 μg/kg BW/day of DiNP on the uterine events critical for the establishment and maintenance of pregnancy in mice.

Successful establishment of pregnancy depends on the timely progression of a series of biological events during which the embryo interacts functionally with the steroid hormone-primed uterus. In mice, implantation is initiated when the embryo attaches to the uterine epithelium on day 4 of pregnancy. As the embryo invades through the luminal epithelium into the underlying stromal compartment, the fibroblastic stromal cells differentiate into secretory decidual cells, a process termed as decidualization [12–14]. The peak of decidualization occurs during days 5 to 7 of gestation. A major function of the decidual tissue is to protect and regulate embryonic growth and development until placentation. The decidua secretes paracrine factors that promote the formation of new blood vessels, ensuring an adequate supply of oxygen and nutrients to the embryo [15, 16]. Proper differentiation of the trophoblast cells, critical for forming a functional placenta, is also influenced by factors, as yet unknown, secreted by the differentiating stromal cells [15–19].

In the present study, pregnant mice were exposed to an environmentally relevant level of DiNP from day 1 to 7 of gestation. This exposure window encompasses both embryo attachment to the uterine epithelium and decidualization, critical events for establishing a successful pregnancy. Our results indicate that DiNP exposure did not impact embryo attachment, but it did cause defective decidualization and impaired development of the angiogenic network in the decidual bed. As pregnancy progressed, we observed dysregulated trophoblast differentiation and defective placental development, causing fetal mortality in DiNP-exposed females.

2. MATERIALS AND METHODS

2.1. Animals

The study is in compliance with ARRIVE guidelines for in vivo studies carried out on animals and all experiments involving animals were conducted in accordance with the National Institutes of Health standards for the use and care of animals. The animal protocols were approved by the University of Illinois Institutional Animal Care and Use Committee. Adult CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA) and allowed to acclimate to the facility before dosing. The mice were housed individually at the College of Veterinary Medicine Animal Facility at the University of Illinois at Urbana-Champaign and provided food and water ad libitum. The temperature at the housing facility was maintained at 22 ± 1 °C with 12-h light-dark cycles to provide a controlled environment.

2.2. Chemicals

DiNP was purchased from Sigma-Aldrich (St. Louis, MO). Tocopherol stripped-corn oil was used as vehicle control and purchased from MP Biomedicals (Solon, Ohio). DiNP purity: >99.5% catalog number 376663-IL, Pcode 102409130. DiNP was diluted in tocopherol-stripped corn oil to obtain a working concentration of 20μg/ml. The volume of 20μg/ml DiNP fed to the mouse was adjusted based on daily body w2.eight gain, to obtain a final in vivo concentration of 20μg/kg BW/day.

2.3. Dosing

CD-1 female mice of 8 weeks were mated with fertile males. Each female mouse was housed with a male until a copulatory plug was observed. The day when the copulatory plug was observed was designated as day 1 of pregnancy, and female mice were randomly assigned to either the control or DiNP group. Each pregnant female was housed individually during the entire duration of this study. Mice, especially pregnant dams, are motivated to construct nests. Therefore, nesting material was provided for cage enrichment. The nesting material facilitates nest building behavior and thermoregulation. Each mouse cage contained a short fiber cotton nestlet which served as a nesting material. The control group received tocopherol-stripped corn oil (vehicle control), while the mice in the DiNP group received 20 μg/kg BW/day dose of DiNP. The dosing volume was calculated and adjusted based on daily body weights, which typically ranged between 28–41μL. The oil or DiNP was orally piped directly into the mouth of the mice from day 1 to day 7 of pregnancy.

2.4. Study design

Pregnant mice were fed corn oil (vehicle control; n=25) or 20 μg/kg BW/day of DiNP (n=25) through days 1 to 7 of gestation. To determine the effect of this exposure on pregnancy outcomes, 7 mice from each of the treatment (control and DiNP) groups were allowed to deliver pubs. The remaining pregnant mice were euthanized, and the implantation chambers were collected on day 7 (n=6), 13 (n=6) and 18 (n=6). Mice were euthanized by CO2 asphyxiation followed by cervical dislocation.

2.5. Blood and Tissue Collection

Mice were euthanized between 9–11 am by CO2 asphyxiation followed by cervical dislocation. Blood was collected immediately after euthanasia by inserting a 26G needle into the heart.

Implantation chambers were collected from the control and DiNP-treated groups on day 7, day 13, and day 18 of pregnancy. For pregnancy day 7, some of the implantation chambers were fixed in 10% neutral buffered formalin (NBF) fixative while the others were flash frozen in liquid nitrogen. For each of the frozen implantation chamber, embryo was dissected out and the decidua was used for RNA isolation. For day 13 and day 18 of pregnancy, the implantation chambers were opened to separate the fetuses from their placentas and weighed. Some placentas were frozen for gene expression analysis, while others were preserved in 10% NBF and processed for histological analysis.

2.6. RNA isolation and gene expression analysis

Total RNA was extracted from decidua and placentas using the Trizol method described previously [16, 19]. For RNA isolation on day 7 of pregnancy, implantation chambers containing the entire decidua but devoid of embryos were used for gene expression analysis. On day 13 and day 18 of pregnancy, gene expression was performed after separating the fetuses from their placentas. RNA was converted to cDNA, and gene expression was performed using quantitative real-time PCR. Real-time quantitative PCR reactions were carried out using SYBR-green master mix (Applied Biosystems) on a QuantStudio™ 3 Real-time PCR instrument (Applied Biosystems). The mean threshold cycle (Ct) for each sample was calculated from Ct values obtained from three replicates. The normalized ΔCt in each sample was calculated as the mean Ct of the target gene subtracted by the mean Ct of the reference gene. ΔΔCt was then calculated as the difference between the ΔCt values of the control and DiNP exposed samples. The fold change of gene expression in each sample relative to control was generated using the 2−ΔΔCt mathematical model for relative quantification of quantitative PCR. The mean fold induction and standard error of the mean (SEM) were calculated from samples collected from all the mice in each treatment group. One sample (uteri or placentas) from each mouse is an independent experimental data point. The primers used for gene expression are listed in Supplementary Table 1.

2.7. Steroid hormone measurements

Steroid hormones, including estrogen and progesterone, were measured using enzyme-linked immunosorbent assays (DRG International Inc., New Jersey) according to the manufacturer’s protocol. A single ELISA experiment was conducted in a 96 well plate where each sample was analyzed in duplicates with a CV range of 0.5– 2.8%.

2.8. Histology

Implantation chambers and placentas were fixed in 10% NBF overnight and dehydrated using a series of increasing concentrations (70% to 100%) of ethanol. Samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

2.9. Area measurements

Area measurements were made in a 5μm mid-placental section obtained from each mouse (n=5) using Adobe photoshop version 23.3.2. The free-hand lasso tool was used to demarcate the labyrinth and the junctional zone. The area measurements were made using ‘record measurements’ tool.

2.10. Immunohistochemistry

Every section from the center of the implantation chambers were selected for hematoxylin and eosin staining and immunofluorescence (IF). For hematoxylin and eosin staining and IF, two implantation chambers from each mouse (N=6) were analyzed. Paraffin-embedded uterine sections were subjected to immunohistochemistry as described previously [15, 16]. Briefly, tissues were deparaffinized in xylene, rehydrated through a graded series of ethanol, and washed in running tap water for 5 minutes. For hematoxylin and eosin staining, a standard procedure was used. For immunostaining for the platelet/endothelial cell adhesion molecule 1 PECAM1 (CD31) staining: Antigen retrieval was performed in a pressure cooker in 10 mM Tri-EDTA buffer (pH 9.0) for 20 min. The tissues were washed between steps (three times for 5 minutes each) using 1x phosphate-buffered saline solution containing 0.05% Tween 20 (PBS-T). Nonspecific binding was inhibited by incubating the sections with 10% normal serum for 1-h at room temperature. After the serum block, sections were incubated overnight at 4 °C with the diluted primary antibody solution [PECAM1 (ab182981; 1:200)] in PBS-T containing 1% normal serum. Labeling was visualized by incubating a fluorescent-tagged secondary antibody for 1-h at room temperature. All incubations were done using a humidified chamber protected from light. Slides were mounted using a mounting solution containing DAPI. Images were taken using a microscope and the OLYMPUS cellSens Standard software (Olympus Corporation, USA).

Quantitation of the immunofluorescence was done using the ImageJ software (https://imagej.nih.gov/ij/) as described previously [20]. Briefly, for each uterine section, fluorescent intensity of the stained area for a single channel was measured using the ‘measure’ tool in the ImageJ software. The corrected fluorescent intensity was obtained by subtracting the background fluorescence from the measured fluorescent intensity of the stained area. The corrected fluorescent intensity was used to calculate the average relative intensity for a uterine section from each of the control and the DiNP-exposed mice (N=6).

2.11. Statistical analyses

Statistical analyses were performed as we have done previously [16]. The normality was tested using QQ plots for t-tests. Briefly, placental and pup weights, gene expression data, hormonal profiles, and relative intensities were expressed as mean ± SEM. Statistical analysis was done using a two-tailed Student’s t-test, Mann-Whitney rank sum test (for single comparison), one-way analysis of variance (ANOVA) with a Bonferroni post-test (for multiple comparisons between samples or time points), or two-way ANOVA with a Bonferroni post-test (for multiple comparisons between different samples and time points). In addition, an analysis of equal variances was done on all numerical data to determine whether a parametric or non-parametric hypothesis test was appropriate. Data were considered statistically significant at p ≤ 0.05. Specific p values are indicated in the figures. All data were analyzed and plotted using GraphPad Prism 9.4 (GraphPad Software).

2.12. Data Availability

All data generated or analyzed during this study are included in this published article.

3. RESULTS

3.1. Exposure to DiNP during early pregnancy affects litter size

During early pregnancy, the uterine environment is particularly vulnerable to chemical exposures, as various cellular components of the uterus and the trophoblast cells of the embryo, which give rise to the placenta, can be functionally altered, leading to pregnancy complications. To test our hypothesis, we designed a study in which pregnant mice were exposed to an environmentally relevant dose (20 μg/kg BW/day) of DiNP through days 1 to 7 of gestation and then determined the effect of this exposure on pregnancy outcomes. Mice fed corn oil (control) produced an average of 16 pups per litter, while mice exposed to DiNP produced an average of 11 pups per litter (Figure 1), which is approximately 30% reduction in litter size on postnatal day 1. The sex ratio of the pups delivered was 1:1.

Figure 1: Exposure to DiNP during early pregnancy affects litter size.

Figure 1:

Open in a new tab

Pregnant mice were fed corn oil (control) or exposed to 20 μg/kg BW/day of DiNP from days 1 to 7 of gestation as described in the Materials and Methods. The number of pups born from control and DiNP-exposed pregnant females were determined at the end of gestation. The data are represented as the mean ± SEM of pups born to each dam. N= 7 dams in each group.

3.2. DiNP exposure impairs differentiation of stromal cells to decidual cells and development of the angiogenic network in the decidual bed

To investigate the cause of reduced litter size in DiNP-exposed females, we analyzed the number of implantation chambers on day 7 of pregnancy. A gross examination of uterine morphology revealed a comparable number of implantation sites in control and the DiNP-treated females (Figure 2A). Histological analysis revealed no apparent defect in embryo attachment in pregnant females exposed to DiNP (Figure 2B). However, when we analyzed the expression of factors critical for decidualization, such as Hand2, Bmp2, Wnt4, and Cebpβ [20–23], we found a significant reduction in their expression in DiNP-exposed uterine tissues compared to controls (Figure 2C). All of these factors are induced downstream of progesterone receptor (PGR) signaling in stromal cells at the onset of decidualization [14, 20–23]. These results indicated that the decidualization process is affected by DiNP exposure during early pregnancy. This defect in decidualization was not due to altered levels of progesterone and estrogen or their cognate receptors PGR1 and estrogen receptor alpha (ESR1), respectively. Serum levels of these hormones and the receptor levels on day 7 of pregnancy did not differ significantly between the controls and DiNP-exposed animals on day 7 of pregnancy (Supplementary Figure 1).

Figure 2. Exposure to DiNP adversely affects stromal cell decidualization.

Figure 2.

Open in a new tab

A. Embryo implantation. The representative images of uteri collected from the control and DiNP-treated group are shown. The average number of implantation chambers in the control and the DiNP- treated mice were 15 on day 7 of pregnancy. B. Histological examination of implantation chambers. Serial sections of uterine horns of control and DiNP-exposed pregnant mice were examined by H&E staining. The representative images from the control and DiNP-treated group are shown. C. Total RNA was purified from DiNP-exposed and unexposed uterine tissues and subjected to qPCR using primers specific for Hand2, Bmp2, Wnt4 and Cebpβ. 36b4 (Rplp0) was used as the internal control. The data are represented as the mean ± SEM where a single data point represents each dam. N= 7 dams in each group.

A primary function of the decidual cells is to secrete angiogenic factors which act on adjacent endothelial cells to promote development of vasculature in the decidual bed. Previous studies have shown that impaired decidualization can adversely impact angiogenesis [15, 16]. We therefore examined the extent of angiogenesis in pregnant uteri of DiNP-exposed mice, employing immunofluorescence using an antibody against PECAM1, a marker of endothelial cells. Uterine sections of the control mice on day 7 of pregnancy exhibited a well-developed vascular network spread in the decidual bed surrounding the implanted embryo. However, exposure to DiNP compromised the development of microvasculature in the mesometrial region of the uterus (Figure 3, left panel). Consistent with this observation, we noted a significant decline in the levels of mRNAs encoding well-known angiogenic regulators, such as vascular endothelial growth factor (Vegf), angiopoietin 2 (Angpt2) and hypoxia inducible factor 2 (Epas1) in the uteri of mice exposed to DiNP. However, DiNP exposure did not alter the expression of angiopoietin 1 (Angpt1) mRNA in the decidua on day 7 of pregnancy (Figure 3, right panel). Taken together, these results indicated that exposure to DiNP during early pregnancy inhibits stromal cell differentiation and, consequently, expression of specific factors that control decidual angiogenesis.

Figure 3. Decidual angiogenesis is impaired upon exposure to DiNP.

Figure 3.

Open in a new tab

Left: Uterine sections of control and DiNP-exposed mice on day 7 of pregnancy were subjected to IF staining with PECAM1 (red) antibody and nuclear stain DAPI (blue). M and E denote mesometrial decidua and embryo, respectively. Magnified images of the boxed areas are shown in the lower panels. ImageJ analysis of PECAM1 positive cells is shown. The values represent mean ± SEM of six independent samples. Right: qPCR was performed to analyze the expression of angiogenic factors, Vegfa, Angpt1, Angpt2, and Epas1 in uteri of control and DiNP-exposed mice on day 7 of pregnancy. The data are represented as the mean ± SEM where a single data point represents each dam. N= 7 dams in each group.

3.3. Mice exposed to DiNP during early pregnancy exhibit altered placental architecture

As pregnancy progresses, a subset of trophoblast cells established during early gestation will form the placenta. To investigate the impact of DiNP exposure on placental development, we analyzed the implantation chambers on day 13 of pregnancy. The number of implantation chambers in the control and the DiNP group was comparable on day 13 (Figure 4A, upper panels). However, the average weights of the fetuses and placentas in the DiNP-exposed females were lower than the average weights of fetuses and placentas from the control group (Figure 4A, lower panels).

Figure 4. Placental architecture is altered in mice exposed to DiNP.

Figure 4.

Figure 4.

Open in a new tab

A. The representative images of the implantation chambers collected from the control and DiNP-treated group on day 13 of pregnancy are shown. While the average number of implantation chambers in the control and the DiNP-treated mice were comparable on day 13 of pregnancy, the weight of the placentas and fetuses in the DiNP group was significantly less than in the control group. B. H & E staining of control and DiNP-exposed placental sections on day 13 of pregnancy are shown. Mid sections from two placenta per mouse were analyzed. The junctional zone has been outlined in both control and DiNP exposed placentas. Lower panels indicate magnified images of the boxed areas. Black arrows indicate the trophoblast cells from the junctional zone that have been mislocalized in the labyrinth. L: labyrinth, JZ: junctional zone, D: decidua. N=6 dams for each group, and representative images are shown. C. Area measurements were performed in a 5μm mid-placental sections obtained from each mouse (N=5) using Adobe photoshop version 23.3.2. The free-hand lasso tool was used to demarcate the labyrinth and the junctional zone. The area measurements were recorded using the ‘record measurements’ tool. The junctional zone area and the total placental area were comparable in the control and DiNP-exposed mice however, the labyrinth area is significantly reduced in the placentas from the DiNP-exposed mice. The ratio of the labyrinth area to the junctional zone area is significantly decreased (p > 0.0001). The data are represented as the mean area ± SEM where a single data point represents a placenta from each dam.

We next investigated the placental architecture on day 13 of pregnancy, using histological analysis of placentas from control and DiNP-exposed mice. As shown in Figure 4B, the placentas of pregnant mice under normal (control) conditions on day 13 consist of three well-organized layers: the labyrinth (L), the junctional zone (JZ) consisting of spongiotrophoblast and glycogen-rich trophoblast cells, and a layer of parietal trophoblast giant cells bordering the maternally derived decidua (D). However, the placentas from DiNP-exposed mice exhibited a disorganized junctional zone, with trophoblast cells from the junctional zone having infiltrated the labyrinth layer (Figure 4B).

Upon closer examination, we found that the total area of the placentas from the DiNP group was slightly but not significantly decreased as compared to the placentas from the control group (Figure 4C). However, average area of the labyrinth was significantly reduced while the junctional zone area was marginally increased in mice exposed to DiNP (Figure 4C). Consequently, the ratio of the labyrinth area to junctional zone in each of the placentas was significantly decreased in the DiNP-exposed mice compared to the unexposed control group (Figure 4C). Collectively, these results indicated that a significant reduction in the labyrinth zone occurs in placentas of mice after exposure to DiNP.

3.4. DiNP exposure during pregnancy affects trophoblast differentiation

Reduction in placental labyrinth often results from impaired trophoblast cell differentiation [24, 25]. To investigate whether trophoblast differentiation is affected by DiNP exposure, we investigated the expression of specific trophoblast subtypes: spongiotrophoblast cells of the junctional zone and syncytiotrophoblast cells (SynT-I and SynT-II) and the sinusoidal trophoblast giant cells, which are major constituents of the labyrinth [17, 18]. As shown in Figure 5A, the expressions of Tpbpa, a specific marker for spongiotrophoblast cells and Ctsq, a specific marker for sinusoidal trophoblast giant cells, were comparable in DiNP-exposed and control unexposed placentas, indicating that the development of these cells was not affected by DiNP treatment. Interestingly, we found a marked decline (p < 0.005) in the expression of SynA, a specific marker for SynT-I cells, and SynB and Gcm1 (glial cell missing-1), factors known to play a critical role in the differentiation of SynT-II syncytiotrophoblast cells of the labyrinth (Figure 5A). Together, these results indicated that DiNP treatment downregulates the expression of SynA, SynB, and Gcm1 in the placentas and, consequently, influences the differentiation of SynT-I and SynT-II cells of the labyrinth layer.

Figure 5. DiNP exposure affects trophoblast differentiation.

Figure 5.

Figure 5.

Open in a new tab

A. Total RNA was isolated from the placenta on day 13 of pregnancy, and qPCR analysis was performed using primers specific for Tpbpa, Ctsq, Gcm1, SynA, and SynB. Data represent mean ± SEM from six separate samples. 36b4 (Rplp0) was used as the internal control. B, Top Panel: A schema showing the expression of glucose transporters, lactate transporters, and connexin 26 in the trophoblast cells of the labyrinth. B, Lower Panel: Total RNA was isolated from the placenta on day 13 of pregnancy, and qPCR analysis was performed using primers specific for Slc2a1 (GLUT1), Slc2a3 (GLUT3), Slc2a4 (GLUT4), Slc6a1 (MCT1), Slc6a4 (MCT4), and Gjb2 (connexin 26). 36b4 (Rplp0) was used as the internal control. Data represent mean ± SEM from six separate dams.

3.5. Attenuated expression of nutrient transporters in placentas of mice exposed to DiNP

Nutrient transfer from a mother to the fetus occurs in the labyrinth compartment of the placenta. In rodents and primates, the labyrinth, demarcated by the syncytiotrophoblast layers, is closely associated with maternal blood spaces, greatly enhancing the surface area across which nutrient transport occurs [26, 27]. In addition, fetal blood vessels and maternal blood are separated by specialized trophoblast cells, which have nutrient transporters and channels that transport the nutrients from the mother to the fetus (Figure 5B, upper panel). Therefore, optimum density of this vascular network and sufficient expression of nutrient transporters and channels are crucial for supplying adequate nutrients to the embryo.

In rodents, nutrient transport from the maternal sinuses occurs across a layer of mononuclear cells lining the sinuses, followed by a bilayer of syncytiotrophoblasts separated by intercellular space (Figure 5B, upper panel). A variety of glucose and lactate transporters localized on these trophoblast cells of the labyrinth facilitate nutrient transport from the mother to the fetus. SLC2A1 (also known as GLUT1) is the primary glucose transporter in the placenta [28]. It is abundantly expressed in the three trophoblast layers of the labyrinth zone as well as in the spongiotrophoblast cells [25, 29]. In addition to SLC2A1, SLC2A3 (GLUT3) and SLC2A4 (GLUT4) are expressed in the mouse placenta [30, 31]. Further, glucose transport between the basal membrane of syncytiotrophoblast layer I and the apical membrane of syncytiotrophoblast layer II is mediated by gap junction channel proteins. Of relevance is the gap junction protein beta 2, Gjb2 or connexin 26. Connexin 26-null mice exhibit a 60% decrease in glucose transport and die at embryonic day 10 [31, 32]. As shown in Figure 5B, lower panel, exposure to DiNP significantly (p < 0.005) downregulated expression of glucose transporters Slc2a1 and Slc2a3 as well as Gjb2 in the placenta while expression of Slc2a4 was not significantly altered upon exposure to DiNP.

In several mammalian species, lactate serves as an alternative energy substrate, especially in conditions with decreased glucose utilization [33]. Solute carrier family 16 members of Slc16 family are proton-coupled monocarboxylate transporters (MCTs) that are essential for transporting lactate, ketone bodies, and other monocarboxylates through the plasma membrane and may contribute to the net transport of lactate through the placental barrier. Slc16a1, also known as MCT1, is localized exclusively on the apical side of syncytiotrophoblast layer I (maternal side), while Slc16a3 or MCT4, is localized exclusively on the basal side of syncytiotrophoblast layer II (fetal side) of the bilayer [33]. Our studies revealed that exposure to DiNP significantly (p < 0.005) downregulated the expression of lactate transporters Slc16a1 and Slc16a3 (Figure 5B, lower panel). These results indicated a significant reduction in the expression of nutrient transporters and channels that are localized in the labyrinth compartment of DiNP-exposed placentas.

3.6. Fetal growth is affected upon exposure to DiNP

Next, we analyzed fetuses from control and DiNP-exposed females towards the end of gestation (Figure 6). By day 18 of pregnancy, the uteri from DiNP-exposed dams showed resorption sites and fewer implantation sites as compared to the controls. The dams exposed to DiNP were observed to have poorly perfused placentas and dead fetuses (circled). While the control dams had healthy fetuses, resorbed fetuses (square) were observed in dams exposed to DiNP. Further, the fetuses collected from DiNP-treated dams weighed significantly less than those in the control group (Figure 6).

Figure 6. Exposure to DiNP during early pregnancy affects fetal growth and mortality.

Figure 6.

Open in a new tab

Pregnant mice were fed corn oil (control) or exposed to 20 μg/kg/day of DiNP from days 1 to 7 of gestation as described in the Materials and Methods. Fetal growth was determined on day 18 of pregnancy. Left: Fetuses and placentas from control dams were healthy. Middle: Dams exposed to DiNP had poorly perfused placentas and resorbed fetuses. At least one resorbed fetus was observed in the DiNP-exposed dams by 18 of pregnancy (N=6). Right: The fetuses from the DiNP-exposed dams weighed significantly less than those from the control dams. The data are represented as the mean number of fetuses ± SEM carried by each dam. N= 6 dams in each group.

We also noted with interest altered gestation length in DiNP-exposed females. While the control mice had a gestation period of 20 days, mice from the DiNP group delivered pups 18–24 hours early, indicating that the timing of parturition is advanced in response to DiNP exposure (Figure 7A). The weight of the fetuses from DiNP-exposed dams remained significantly lower than that of control dams at postnatal day 1 (Figure 7B). Collectively, these results indicated that DiNP exposure during early pregnancy interferes with trophoblast differentiation, causing a defect in placentation, potentially compromising proper nutrient transport to the fetus, and causing fetal morbidity and mortality.

Figure 7. Exposure to DiNP affects gestation length and fetal growth.

Figure 7.

Open in a new tab

A: Pregnant mice exposed to corn oil had a gestation length of 20 days, while mice exposed to DiNP delivered pups 20–24 hours early, resulting in a gestation period of 19 days. B: Pup weights were measured on postnatal day 1. Pups from the DiNP-exposed dams weighed significantly less than those from the control dams. The data are represented as the mean ± SEM of pups born to each dam. N= 7 dams in each group.

4. DISCUSSION

In the past few years, DiNP has been steadily replacing a structurally similar phthalate, di(2-ethylhexyl) phthalate (DEHP), in consumer products, mainly because of increasing awareness about DEHP toxicity. Previous studies showed that exposure to DEHP reduces embryo implantation, reduces placental growth, and increases embryonic loss in mice. However, these studies used high doses, far exceeding environmental exposure levels [34, 35]. Interestingly, our previous study showed that exposing pregnant mice to an environmentally relevant level of DEHP did not impact uterine functions unless the mice were fed a high fat diet [25]. Furthermore, no significant difference in the number of implantation sites, placental growth and architecture, or litter size was observed in response to DEHP exposure [25]. In contrast, in the present study, we report that exposure to an environmentally relevant level of DiNP led to a defect in placenta development and caused fetal morbidity and mortality. These results raise the possibility that DiNP may be more toxic than DEHP, at least in the context of mouse pregnancy.

Our study revealed that exposure to DiNP affects differentiation of stromal cells into decidual cells during early pregnancy. Differentiation of endometrial stromal cells into decidual cells is accompanied by development of vascular networks in the decidua and, indeed, decidual angiogenesis was impacted by exposure to DiNP. Previous studies indicated that the secretion of angiogenic factors such as VEGFA, ANGPT1, and ANGPT2 from the decidual cells stimulates vasculature development in the decidual bed [36, 37]. VEGFA stimulates de novo vasculogenesis [38] and is the major driver of decidual angiogenesis [37], while the role of ANGPT2 during early pregnancy is to initiate branching of blood vessels followed by their elongation and sprouting, which leads to the formation of a complex vascular network [39]. Studies show that women with miscarriages have lower expression of VEGFA and its receptors [40]. Additionally, the decidual tissues of women with recurrent miscarriages have lower angiopoietin 2 expression than those with normal pregnancies [41].

In this study, we report that DiNP exposure decreases the expression of Vegfa and Angpt2 in the decidua, thereby compromising the development of vasculature during early pregnancy. A previous report indicated that Vegfa expression during decidualization is regulated primarily by PGR [37]. We also show that, along with Vegfa and Angpt2, the expressions of Cebpβ, Hand2, and Bmp2 are attenuated in response to DiNP exposure. Notably, Cebpβ, Hand2, and Bmp2 are critical factors for decidualization and are regulated by PGR signaling in endometrial stromal cells [20–23]. Furthermore, P/PGR signaling regulates the expression of other angiogenic factors, such as Angpt2, during early pregnancy [36]. Based on these results, it is conceivable that DiNP exposure during early pregnancy interferes with PGR-signaling in the uterus. Interestingly, DiNP exposure did not affect the production of progesterone during early pregnancy (day 7; Supplementary Figure 1) and mid pregnancy (day 13; data not shown). Although the ovarian histology including the number of corpora lutea were not evaluated, the serum levels of progesterone estimated using ELISA were comparable between the control and the DiNP exposed dam, suggesting that the ovarian function required to maintain pregnancy was intact. Further studies are required to completely understand how DiNP disrupts PGR-signaling in the uterus.

Continued exposure to DiNP from day 1 to day 7 of pregnancy in mice also impacts the differentiating trophoblast precursor cells involved in placentation. On day 5 of pregnancy, the mural trophectoderm cells differentiate into the extraembryonic ectoderm and the ectoplacental cone. As gestation progresses, the cells from the ectoplacental cone and extraembryonic ectoderm lineages differentiate into the spongiotrophoblast cells of the junctional zone and trophoblast cells of the labyrinth, respectively [17, 24]. Our studies revealed that the expression of Tpbpa, a marker of spongiotrophoblast cells residing in the junctional zone of the placenta, is comparable in the placentas of unexposed and DiNP-exposed mice. However, the expressions of SynA, SynB, and Gcm1, which play an important role in trophoblast differentiation and development of the labyrinth layer of the placenta, were significantly reduced in DiNP-exposed placentas. These results indicated that exposure to DiNP compromises the programmed differentiation of trophoblast cells in the labyrinth. Indeed, abnormal differentiation and function of the trophoblasts are the basis of many placenta-based pregnancy disorders, including fetal growth restriction [24, 25]. It is, therefore, not surprising that a significant reduction in fetal weight was observed when mice were exposed to DiNP. Although there are considerable differences in placental development between mice and humans, the nutrient exchange pathway involving syncytiotrophoblast is similar in both species [42].

In placental mammals, the major determinant of intrauterine growth is nutrient delivery to the fetus via the placental labyrinth, which occurs primarily by diffusion and transporter-mediated mechanisms. Transport of nutrients in the placenta depends on the size, morphology, and blood flow of the labyrinth. Therefore, aberrant labyrinth development is associated with impaired intrauterine growth. Typically, the labyrinth region comprises about 75% of the total placenta area, while the junctional zone spans the remaining 25%. Typically, wild-type mice, placentas that weigh less during mid pregnancy often have a reduced decidua and/or junctional zone but no significant difference in the labyrinth area as a result the fetus growth is not compromised [43]. However, in our study we observed that in the DiNP-exposed placentas, the labyrinth area in proportion to the total area of the placenta was reduced by approximately 20%, thereby decreasing the surface area available for nutrient exchange. Branching of the blood vessels or the vascular density did not change significantly between the control and the DiNP placentas indicating that compensation due to smaller labyrinth did not occur in the DiNP group. Instead, we observed a significant decline in the expression of nutrient transporters located on the trophoblast cells in the labyrinth of mice exposed to DiNP. This deficiency in transporter expression is likely to dampen the nutrient transfer from mother to fetus, affecting fetal growth.

Placentas from both sexes were used in the study. While placentas of male fetuses weigh more than the placentas of female fetuses, the ratio of the labyrinth to junctional zone areas is unaffected by sex ensuring optimum nutrient transfer irrespective of the sex [44]. Therefore, the decline in the ratio of the labyrinth to junctional zone areas observed in the DiNP-exposed females is not confounded by the sex of the fetuses. Further, we observed that the average weight of the placentas of male and female fetuses from the DiNP-exposed females was less than the average weight of the placentas of male and female fetuses from the control females. To the best of our knowledge, the expression of genes that were down regulated in the placentas of the DiNP-exposed females have not been reported to show sexual dimorphism. Therefore, the impact of DiNP exposure on placentation reported in this study appears independent of the sex of the fetus.

Mice exposed to DiNP lost 30% of their pups towards the end of gestation. While placental and fetal weights were lower in DiNP-exposed pregnant females at mid-gestation on day 13 compared to controls, the number of the implantation sites were comparable between control and the DiNP exposed and resorption sites indicating fetal loss were not observed at that time. Therefore, there was no post implantation loss up to day 13 of gestation. However, on day 18, DiNP-exposed pregnant females exhibited resorption sites and delivered pups several hours earlier than unexposed controls. Every mouse from the DiNP group had at least 2 resorption sites in addition to poorly perfused placentas and dead fetuses inside the implantation chambers. The underlying mechanisms by which DiNP influences the timing of parturition remain unknown. Likewise, it is unclear how DiNP affects molecular pathways involved in the differentiation of specific trophoblast lineages during placentation. Future studies will employ single-cell RNA sequencing and bioinformatic analysis to elucidate the underlying mechanisms by which DiNP impacts placental development.

In summary, exposure to DiNP causes fetal loss towards the end of gestation. Furthermore, the pups born to DiNP-exposed pregnant females weighed less than the pups born to the control dam. Significant decline in the expression of nutrient transporters located on the trophoblast cells in the labyrinth of mice exposed to DiNP is likely to dampen the nutrient transfer from mother to fetus, affecting fetal growth. Although a single dose toxicity study is not ideal, there are interesting data presented concerning the outcomes from use of this dose. Future studies will be aimed at dissection of these outcomes using multiple doses.

Supplementary Material

1
2
3
4

Highlights.

  • DiNP exposure impacts uterine angiogenesis

  • DiNP exposure affects placentation

  • DiNP exposure affects fetal growth

Acknowledgments

This work was supported by NIH (R01 ES032163 and T32 ES007326).

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Disclosure Statement:

The authors have nothing to disclose.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  • 1.CPSC USCPSC, Chronic Hazard Advisory Panel On Diisononyl Phthalate (DiNP). 2001. [Google Scholar]
  • 2.Chap, Chronic Hazard Advisory Panel on phthalates and phthalate alternatives (with appendices). 2014, U.S. Consumer Product Safety Commission, Directorate for Health Sciences: Bethesda, MD. [Google Scholar]
  • 3.Fromme H, et al. , Phthalates and their metabolites in breast milk--results from the Bavarian Monitoring of Breast Milk (BAMBI). Environ Int, 2011. 37(4): p. 715–22. [DOI] [PubMed] [Google Scholar]
  • 4.Frederiksen H, Jørgensen N, and Andersson AM, Correlations between phthalate metabolites in urine, serum, and seminal plasma from young Danish men determined by isotope dilution liquid chromatography tandem mass spectrometry. J Anal Toxicol, 2010. 34(7): p. 400–10. [DOI] [PubMed] [Google Scholar]
  • 5.Toft G, et al. , Association between pregnancy loss and urinary phthalate levels around the time of conception. Environ Health Perspect, 2012. 120(3): p. 458–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Hines CJ, et al. , Occupational exposure to diisononyl phthalate (DiNP) in polyvinyl chloride processing operations. Int Arch Occup Environ Health, 2012. 85(3): p. 317–25. [DOI] [PubMed] [Google Scholar]
  • 7.Minguez-Alarcon L, et al. , Urinary concentrations of bisphenol A, parabens and phthalate metabolite mixtures in relation to reproductive success among women undergoing in vitro fertilization. Environ Int, 2019. 126: p. 355–362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Specht IO, et al. , Serum phthalate levels and time to pregnancy in couples from Greenland, Poland and Ukraine. PLoS One, 2015. 10(3): p. e0120070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Smarr MM, et al. , Parental urinary biomarkers of preconception exposure to bisphenol A and phthalates in relation to birth outcomes. Environ Health, 2015. 14: p. 73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Chiang C and Flaws JA, Subchronic Exposure to Di(2-ethylhexyl) Phthalate and Diisononyl Phthalate During Adulthood Has Immediate and Long-Term Reproductive Consequences in Female Mice. Toxicol Sci, 2019. 168(2): p. 620–631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chiang C, et al. , Exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate during adulthood disrupts hormones and ovarian folliculogenesis throughout the prime reproductive life of the mouse. Toxicol Appl Pharmacol, 2020. 393: p. 114952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Cha J, Sun X, and Dey SK, Mechanisms of implantation: strategies for successful pregnancy. Nat Med, 2012. 18(12): p. 1754–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Pawar S, et al. , Uterine Epithelial Estrogen Receptor-alpha Controls Decidualization via a Paracrine Mechanism. Mol Endocrinol, 2015. 29(9): p. 1362–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Bhurke AS, Bagchi IC, and Bagchi MK, Progesterone-Regulated Endometrial Factors Controlling Implantation. Am J Reprod Immunol, 2016. 75(3): p. 237–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Bhurke A, et al. , A hypoxia-induced Rab pathway regulates embryo implantation by controlled trafficking of secretory granules. Proc Natl Acad Sci U S A, 2020. 117(25): p. 14532–14542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Davila J, et al. , Rac1 Regulates Endometrial Secretory Function to Control Placental Development. PLoS Genet, 2015. 11(8): p. e1005458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Cross JC, How to make a placenta: mechanisms of trophoblast cell differentiation in mice--a review. Placenta, 2005. 26 Suppl A: p. S3–9. [DOI] [PubMed] [Google Scholar]
  • 18.Cross JC, Werb Z, and Fisher SJ, Implantation and the placenta: key pieces of the development puzzle. Science, 1994. 266(5190): p. 1508–18. [DOI] [PubMed] [Google Scholar]
  • 19.Bhurke A, Bagchi MK, and Bagchi IC, Uterus: Growth Factors and Cytokines, in Encyclopedia of Reproduction (Second Edition), Skinner MK, Editor. 2018, Academic Press: Oxford. p. 333–338. [Google Scholar]
  • 20.Lee KY, et al. , Bmp2 is critical for the murine uterine decidual response. Mol Cell Biol, 2007. 27(15): p. 5468–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Li Q, et al. , WNT4 acts downstream of BMP2 and functions via beta-catenin signaling pathway to regulate human endometrial stromal cell differentiation. Endocrinology, 2013. 154(1): p. 446–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Li Q, et al. , The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2. Science, 2011. 331(6019): p. 912–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Mantena SR, et al. , C/EBPbeta is a critical mediator of steroid hormone-regulated cell proliferation and differentiation in the uterine epithelium and stroma. Proc Natl Acad Sci U S A, 2006. 103(6): p. 1870–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kannan A, et al. , Maternal high-fat diet during pregnancy with concurrent phthalate exposure leads to abnormal placentation. Sci Rep, 2021. 11(1): p. 16602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Woods L, Perez-Garcia V, and Hemberger M, Regulation of Placental Development and Its Impact on Fetal Growth-New Insights From Mouse Models. Front Endocrinol (Lausanne), 2018. 9: p. 570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Watson ED and Cross JC, Development of structures and transport functions in the mouse placenta. Physiology (Bethesda), 2005. 20: p. 180–93. [DOI] [PubMed] [Google Scholar]
  • 27.Coan PM, Ferguson-Smith AC, and Burton GJ, Developmental dynamics of the definitive mouse placenta assessed by stereology. Biol Reprod, 2004. 70(6): p. 1806–13. [DOI] [PubMed] [Google Scholar]
  • 28.Winterhager E and Gellhaus A, Transplacental Nutrient Transport Mechanisms of Intrauterine Growth Restriction in Rodent Models and Humans. Front Physiol, 2017. 8: p. 951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Wang D, et al. , A mouse model for Glut-1 haploinsufficiency. Hum Mol Genet, 2006. 15(7): p. 1169–79. [DOI] [PubMed] [Google Scholar]
  • 30.Carruthers A, et al. , Will the original glucose transporter isoform please stand up! Am J Physiol Endocrinol Metab, 2009. 297(4): p. E836–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Ganguly A, et al. , Glucose transporter isoform-3 mutations cause early pregnancy loss and fetal growth restriction. Am J Physiol Endocrinol Metab, 2007. 292(5): p. E1241–55. [DOI] [PubMed] [Google Scholar]
  • 32.Gabriel HD, et al. , Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice. J Cell Biol, 1998. 140(6): p. 1453–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Nagai A, et al. , Cellular expression of the monocarboxylate transporter (MCT) family in the placenta of mice. Placenta, 2010. 31(2): p. 126–33. [DOI] [PubMed] [Google Scholar]
  • 34.Li R, et al. , Effects of DEHP on endometrial receptivity and embryo implantation in pregnant mice. J Hazard Mater, 2012. 241–242: p. 231–40. [DOI] [PubMed] [Google Scholar]
  • 35.Yu Z, et al. , Gestational di-(2-ethylhexyl) phthalate exposure causes fetal intrauterine growth restriction through disturbing placental thyroid hormone receptor signaling. Toxicol Lett, 2018. 294: p. 1–10. [DOI] [PubMed] [Google Scholar]
  • 36.Park YG, Choi J, and Seol JW, Angiopoietin-2 regulated by progesterone induces uterine vascular remodeling during pregnancy. Mol Med Rep, 2020. 22(2): p. 1235–1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kim M, et al. , VEGF-A regulated by progesterone governs uterine angiogenesis and vascular remodelling during pregnancy. EMBO Mol Med, 2013. 5(9): p. 1415–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Ancelin M, et al. , A dynamic shift of VEGF isoforms with a transient and selective progesterone-induced expression of VEGF189 regulates angiogenesis and vascular permeability in human uterus. Proc Natl Acad Sci U S A, 2002. 99(9): p. 6023–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Fagiani E and Christofori G, Angiopoietins in angiogenesis. Cancer Lett, 2013. 328(1): p. 18–26. [DOI] [PubMed] [Google Scholar]
  • 40.Vuorela P, et al. , VEGF, its receptors and the tie receptors in recurrent miscarriage. Mol Hum Reprod, 2000. 6(3): p. 276–82. [DOI] [PubMed] [Google Scholar]
  • 41.Plaisier M, et al. , Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions. Hum Reprod, 2009. 24(1): p. 185–97. [DOI] [PubMed] [Google Scholar]
  • 42.Takahashi K, Kobayashi T, and Kanayama N, p57(Kip2) regulates the proper development of labyrinthine and spongiotrophoblasts. Mol Hum Reprod, 2000. 6(11): p. 1019–25. [DOI] [PubMed] [Google Scholar]
  • 43.Albers RE, et al. , Gestational differences in murine placenta: Glycolytic metabolism and pregnancy parameters. Theriogenology, 2018. 107: p. 115–126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kaur H, et al. , A sexually dimorphic murine model of IUGR induced by embryo transfer. Reproduction, 2021. 161(2): p. 135–144. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1923019-supplement-1.pdf (290.9KB, pdf)
2
NIHMS1923019-supplement-2.docx (27.1KB, docx)
3
NIHMS1923019-supplement-3.docx (34.3KB, docx)
4
NIHMS1923019-supplement-4.docx (34.2KB, docx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

RESOURCES