Oral exposure to di(2-ethylhexyl) phthalate alters the expression of GATA6, OCT4, and CDX2 in blastocysts and decreases the rate of implantation in a mouse model.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer ubiquitously found in the environment. Due to its biological activity, it is classified as an endocrine-disrupting chemical and reproductive toxicant. DEHP and its metabolites have been detected in women with various infertility-related pathologies, and their concentrations have been associated with reduced embryo quantity and quality, implantation failure, and miscarriage in humans. The formation of the inner cell mass and trophectoderm in blastocysts is a critical fate decision for continued development and cellular differentiation, accompanied by the expression of GATA6, OCT4, and CDX2. This study tested whether DEHP induces deleterious conformational changes in blastocysts, potentially leading to reduced implantation rates. Adult female CD-1 mice were exposed to vehicle (corn oil) or DEHP (0, 20, 200, or 2,000 μg/kg/day) orally for 1 mo. The 2,000 μg/kg/day dose induced oocyte and embryo fragmentation. Embryo developmental arrest was evident at DEHP doses of 200 and 2,000 μg/kg/day. DEHP affected the levels and expression patterns of GATA6, OCT4, and CDX2 at doses of 200 and 2,000 μg/kg/day. These doses also impacted the number and functionality of blastocysts. Furthermore, DEHP doses of 200 and 2,000 μg/kg/day impaired endometrial implantation capacity, as evidenced by the failure to implant normal blastocysts from untreated females using transcervical embryo transfer. Collectively, these data suggest that oral exposure to DEHP for 1 mo affects the expression of GATA6, OCT4, and CDX2, consequently reducing implantation capacity.

DEHP, embryo, blastocyst, GATA6, OCT4, CDX2, embryo implantation Graphical abstract Phthalate esters, including di(2-ethylhexyl) phthalate (DEHP), are commonly used as plasticizers in polyvinyl chloride (PVC) modification. DEHP is one of the most widely used phthalates in various applications, with its concentration in final PVC formulations reaching up to 50% (Morgan and Mukhopadhyay 2022). DEHP is released from the polymeric matrix over time, as it is not chemically bound, and subsequently migrates into liquids, foodstuffs (Fang et al. 2017), and various environmental sources due to decomposition and leaching processes, resulting in ubiquitous exposure (Net et al. 2015; Cui et al. 2022; Zhong et al. 2023). Due to its environmental persistence, DEHP is listed among the 6 phthalate esters (PAEs); its chemical stability confers low water solubility, making it difficult to degrade in ecological matrices such as sediments, aquatic environments, and biotic factors (Shivaraju et al. 2021; Zhu et al. 2022). Although humans and animals can rapidly metabolize DEHP, bioaccumulation and biomagnification occur when the rate of excretion is lower than the rate of accumulation (He et al. 2020; Liao et al. 2023), These phenomena can be exacerbated by prolonged exposure over time due to the high concentration of plasticizers like DEHP in the environment (Chormare and Kumar 2022; Liu et al. 2024). This is primarily driven by the production of plastics, which, according to The Organization for Economic Cooperation and Development (OECD 2022), doubled between 2000 and 2019. Additionally, it has been reported that ∼22% of plastics are disposed of in an uncontrolled manner into the environment. Oral exposure to DEHP is considered the primary route of exposure in humans (da Costa et al. 2023). In a controlled study, the daily oral intake for humans weighing 60 kg was determined to be 3.25 μg/kg/day (Sui et al. 2014) and in a study involving pregnant women, total dietary intake ranged from 0.41 to 2.01 μg/kg/day, with dairy products and canned foods being the main sources of exposure (Martínez et al. 2017). The European Food Safety Authority (EFSA CEP Panel 2019) has set the tolerable daily intake at 50 μg/kg/day, whereas the no observed adverse effect level is 4.8 mg/kg/day (Wang and Qian 2021). In the United States, the maximum exposure dose of DEHP ranges from 1 to 30 μg/kg/day (Agency for Toxic Substances and Disease Registry 2022). However, one study determined that the average intake for adults is 0.673 μg/kg/day (Schecter et al. 2013). Based on its biological activity, DEHP exhibits endocrine-disrupting properties, as it can interfere with hormones related to reproductive functions (Ernst et al. 2020; Moche et al. 2021). Low doses of DEHP cause reproductive effects in animal models (Tassinari et al. 2021; Wang et al. 2024) and it is classified as a possible carcinogen (Lunn et al. 2022; Yang et al. 2024). The study of low-dose DEHP exposure remains relevant as this compound and its metabolites continue to be detected in populations and occupational biomonitoring (Fréry et al. 2020; Janjani et al. 2024). In women, quantified levels of DEHP and its metabolites associate with gestational losses (Nobles et al. 2023), histopathological changes in the uterus (Cheon 2020), alterations in folliculogenesis (Feng et al. 2024), infertility (Li et al. 2024a), and transgenerational epigenetic effects (Van Cauwenbergh et al. 2020). The mechanism of action remains unclear; however, studies have reported that DEHP and its metabolites reduced litter sizes in animal models (Rattan et al. 2018; Adam et al. 2022), decreased numbers of oocytes and embryos (Deng et al. 2020), and led ultimately, to embryonic resorption and conceptus loss (Zhang et al. 2020; Aimuzi et al. 2022). Recurrent pregnancy loss affects ∼10% of patients undergoing assisted reproduction treatments. In the general population, it is estimated to impact around 5% of individuals (Raut and Raut 2022). Following fertilization, the embryo undergoes continuous division, progressing through the stages of the 2-cell embryo, multicellular morula, and blastocyst (Rossant and Tam 2022). The blastocyst develops through continuous cell division and differentiation, leading to the formation of the inner cell mass (ICM) and the trophectoderm (TE) (Marsico et al. 2023; McCoy et al. 2023; Skory 2024). This morphological separation is regulated at the molecular level by the expression of GATA-binding factor 6 (GATA6) and octamer-binding transcription factor 4 (OCT4) in the ICM, and by the expression of the homeobox protein CDX-2 (CDX2) in the TE (Yang et al. 2020; Rosner and Hengstschläger 2024). These molecules regulate transcription and epigenetic modifications necessary for epiblast formation from the ICM and for trophoblast formation in the TE. (Sivanantham et al. 2022; Latham 2024). These structures are responsible for initiating embryonic organogenesis and forming extraembryonic tissues, essential for uterine implantation (Weberling and Zernicka-Goetz 2021). Thus, GATA6, OCT4, and CDX2 are crucial for progressing and regulating blastocyst development (Toyooka 2020; Han et al. 2022; Geiselmann et al. 2025). DEHP exposure has been reported to decrease embryo development rates in mice, reducing the blastocyst formation rate in both in vivo and in vitro experiments (Parra-Forero et al. 2019; Arcanjo et al. 2023). Although the molecular machinery involving embryo and blastocyst development has been extensively studied, it remains unclear whether DEHP exposure impairs embryo development by interfering with CDX2, GATA6, and OCT4. Thus, our objective was to assess the functionality of blastocysts from DEHP-exposed mice and correlate this with the expression of transcription factors (GATA6, OCT4, and CDX2) crucial for developmental progression and future implantation.

Reagents DEHP (99% purity), cell culture phosphate-buffered saline (No. MT21040CV), bovine serum albumin, heat shock treated (No.BP1600-100), paraformaldehyde, 4% in PBS (No. J61899.AP) were obtained from Sigma-Aldrich (St Louis, MO). Tocopherol-stripped corn oil (MP Biomedicals, Solon, OH) was used as the vehicle control. Pregnant mare serum gonadotropin (PMSG) was purchased from Prospec (No. HOR-272) and Intervet (Folligon), human chorionic gonadotropin (hCG) was purchased from MilliporeSigma (No. C1063). Isofluorane was purchased from PisaAgropecuaria (Sofloran). Animals and dosing This study was conducted in a multicenter manner at the Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN) (Mexico City, Mexico) and the University of Illinois (Urbana-Champaign, United States). Complete experiments were carried out at each site and analyzed separately. The females were exposed to a subchronic repeated-dose toxicity schedule for 1 month (Denny and Stewart 2024). The groups and concentrations evaluated were as follows: Control group (Oil only), DEHP 20 μg/kg/day, DEHP 200 μg/kg/day, and DEHP 2,000 μg/kg/day. At Cinvestav-IPN, female CD-1 mice of 25 days of age, fed with a rodent diet (Formulab 5008; LabDiet, Brentwood, MO, United States), were provided by the Unit for Production and Experimentation of Laboratory Animals (UPEAL) of Cinvestav-IPN. Female mice were acclimatized for a 2-wk period and dosing with DEHP was started at 40 days of age. During exposure to DEHP, the animals were housed in polysulfonate boxes on a 12 h light/dark cycle and fed ad libitum food and water purified by reverse osmosis. All treatment groups had 5 to 6 animals each. The procedures on experimental animals were performed according to those established for the use and care of laboratory animals and according to protocol 0147-15 approved by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) of Cinvestav-IPN. At University of Illinois Urbana-Champaign, adult female CD-1 mice (25 days) were purchased from Charles River Laboratories (Wilmington, MA) and housed at the University of Illinois Urbana-Champaign College of Veterinary Medicine facility (Urbana, IL). The cages contained 1/8 corn cob bedding (Shepherd Specialty Papers), and environmental enrichment (iso-BLOX, Cat. No. 6060; Envigo). The animals were provided food and water purified by reverse osmosis ad libitum and housed in a controlled animal room environment, maintained at a temperature of 22 ± 1 °C and light-dark cycles of 12 h. Mice were acclimated for 2 wk and put on the dosing study at 40 days of age. Females were divided into groups as follows (control, n = 11; DEHP 20 μg/kg/day, n = 10; DEHP 200 μg/kg/day, n = 10; DEHP 2,000 μg/kg/day, n = 12). The females were exposed to this subchronic repeated-dosing schedule for 1 month (Denny and Stewart 2024). The University of Illinois Institutional Committee approved procedures and housing conditions. The same DEHP reagents and oil were used in both labs. DEHP solutions were prepared using corn oil stock solutions with concentrations of 0.0125, 0.125, and 1.25 mg/ml. To make the final concentrations for administration, weekly preparations were made so that each of the concentrations remained at 20, 200, and 2,000 μg/μl. The animals (40 days old) were weighed daily and the amount of DEHP administered was corrected for weight gain. Administration was by direct deposition in the female’s mouth. All solutions were stored in a cool place protected from light (Richardson et al. 2018). Body weight was recorded, and the estrous cycle was monitored daily through vaginal cytology, adhering to the criteria established by Cora et al. (2015). Experiment 1 Blastocyst recovery To obtain blastocysts, females were subjected to a superovulation protocol with 6 IU of PMSG and then 48 h later they received an injection of 6 IU of hCG. Immediately after the hCG injection, the females were placed with fertility-tested CD-1 males overnight in a male: Female ratio of 1:1 or 1:2, the next day the males were removed. Then, on day 4.5 the females were euthanized, and the union of the ovary, the oviduct, and the uterus of both horns were obtained by abdominal dissection and collected in holding medium (PBS+BSA 0.5%) at a temperature of ∼35 °C. Using a stereoscope at 37 °C, the oviduct, and uterine horns were separated using 35 mm dishes and perfused with PBS using an insulin syringe with a 30-G blunt needle into a new dish. The presumptive blastocysts were retrieved by aspiration with a glass pipette (see Fig. 1A). Evaluation of blastocyst development The blastocysts obtained were quantified at the different observed stages of development. The Gardner Grading Scale criteria were used to evaluate blastocysts based on the degree of expansion (Balaban and Gardner 2013). The categories used in this study were: (i) Early; blastocyst with blastocoel size less than half the embryo volume. (ii) Late blastocyst; blastocyst with a blastocoel equal to or larger than half the embryo volume, and (iii) Expanded blastocyst with a blastocoel volume larger than the early embryo, with a thinned zona pellucida. The evaluation of blastocyst quality was performed using the global grade, which is a subjective evaluation based on specific morphological characteristics to determine the quality of the ICM, considering 3 categories: (i) Good: ICM with a large number of cells organized in a single organized and uniform structure. (ii) Fair: ICM with a moderate number of cells with disorganized areas, losing uniformity. (iii) Poor: ICM without identification, disorganized cells without uniformity. Similarly, the TE was evaluated using the following categories: (i) Good: The TE presents a differentiated, continuous, uniform, and organized cell layer. (ii) Fair: TE with cells with moderate differences in size and shape, but continuous, uniform, and organized. (iii) Poor: TE with few cells and interruption of the continuity of the layer. This experiment was conducted at the Cinvestav-IPN facilities following the protocol outlined in “Blastocyst recovery” section. Experiment 2 Rate of embryo fragmentation and cellular arrest During blastocyst recovery, embryos were identified at various stages of development: 1-cell, 2-cell, and morula. These were quantified and classified as embryos in cell arrest. Additionally, embryos exhibiting fragmentation were quantified. The fragmentation index and cell arrest rate were calculated using the following formulas: For each count, it was ensured that 2 trained persons reviewed the counts and the morphologic quality until a common agreement was reached at each research center. Evaluation by embryo immunostaining of transcription factors in ICM and TE Immunofluorescence was performed on blastocysts immediately after euthanasia. Uteri and oviducts were handled using M2 medium (Sigma-Aldrich, Cat. No. M7167). Washing was conducted by inserting a 33G×8 mm hypodermic needle and perfusing with 0.5 ml of EmbryoMax Advanced KSOM Embryo Medium (Sigma-Aldrich, Cat. No. MR-101). The media were maintained at 37 °C, and the retrieval was performed using a stereoscope with a thermal plate, maintaining the same temperature. Blastocysts were collected using a glass pipette and incubated with 4% PFA for 30 min, followed by 5% Triton X-100 for 15 min, and then 1% BSA for 2 h. Primary antibody incubation was carried out overnight at 4 °C. The primary antibodies used were: Human GATA 6, Polyclonal Goat IgG (R&D Systems, Cat. No. AF1700), Oct-4A (D6C8T) rabbit mAb (Cell Signaling, Cat. No. D6C8T), and anti-CDX2 mouse monoclonal (Biogenex, Cat. No. MU392A) all diluted 1:300. Secondary antibodies used were Cy5 AffiniPure Donkey Anti-Mouse IgG (H + L) (690 nm), Cy3 AffiniPure Donkey Anti-Rabbit IgG (H + L), and Alexa Fluor 488 AffiniPure Donkey Anti-Goat IgG (H + L), all diluted 1:800, for 1 h at room temperature. DAPI (Sigma-Aldrich, Fluoroshield, Cat. No. F6182) was used to stain the DNA of blastocysts during a 15-min incubation at room temperature. Samples were mounted on slides and covered with coverslips. Preparations were stored at −20 °C until analysis with a confocal microscope. Immunostained blastocysts were imaged at the central facilities of the Carl R. Woese Institute for Genomic Biology. Images were acquired with a confocal microscope (Zeiss LSM 880) using the Plan-Apochromat 20×/0.8 objective, scan mode z-stacks, and optical sections were obtained every 6 to 8 μm. The samples were excited with 405, 488, 561, and 633 nm laser wavelengths. Fluorescence intensity was measured using the profiling tool of ZEN 2.3 Lite software. Intensity levels were reported in arbitrary fluorescence units (AFUs) (Hoshino et al. 2023). We conducted the counting of GATA6, OCT4, and CDX2 positive cells to elucidate the spatial patterns within the blastocyst, driven by the competitive expression that occurs during the transition between developmental stages. The cell counting was performed manually using 3D images of each blastocyst, constructed with the ZEN 2.3 Lite software. An image depicting a representative stack exemplifying the ICM site of negative controls for the antibodies used in the immunofluorescence assays is available in Figs S1–S3. This experiment was conducted at the University of Illinois Urbana-Champaign facilities. Experiment 3 Evaluation of implantation To evaluate the implantation capacity of DEHP-exposed blastocysts, we designed the following experiment using both exposed and unexposed uteri to obtain quantitative data. Eight groups, each consisting of 6 animals, were formed and divided into 4 categories: (i) Donor animals with DEHP exposure (0, 20, 200, and 2,000 μg/kg/day), (ii) Donor animals without DEHP exposure (vehicle only), (iii) Recipient animals with DEHP exposure (0, 20, 200, and 2,000 μg/kg/day), and (iv) Recipient animals without DEHP exposure (vehicle only). Blastocyst implantation rates were assessed in 2 schemes: Experiment (3A) blastocysts from DEHP-exposed females were transferred into vehicle (corn oil) exposed females, and Experiment (3B) blastocysts from vehicle (corn oil)-exposed females were transferred into DEHP-exposed recipient females. Blastocysts were obtained from CD-1 donor female mice (8 to 9 wk). Donor mice were injected with 6 IU of PMSG and 48 h later they received an injection of 6 IU of hCG, immediately after which mating was performed. Blastocyst harvest was performed on day 4.5 after mating. The same dissection protocol was followed with M2 medium (Sigma Aldrich, No. M7167). Embryo transfer Female CD1 mice were used as recipients, followed daily during their estrous cycle, and 48 h after day 1 of estrus were mated with vasectomized CD-1 males to ensure pseudopregnancy. On day 2.5 after mating, transcervical transfer was performed, following the protocol described by (Cui et al. 2014). The surgical anesthesia protocol consisted of induction in a chamber with 2% to 3% isoflurane at a flow rate of 0.8 to 1.0 l/min; maintenance was performed with 1.5% to 2% at a flow rate of 0.4 to 0.8 liters/min. Twenty expanded blastocysts with good-quality ICM and TE were transferred into each female mouse recipient. At the end of the transfer, the females were moved to recovery in a chamber with room temperature at 24 °C to 28 °C and monitored until the complete reestablishment of consciousness and normal mobility. For the evaluation of the implantation of the transferred embryos, 1% Chicago Sky Blue was injected intravenously through the coccygeal vein on day 2.5 posttransfer. The same euthanasia and abdominal surgical dissection procedure was followed as described for the previous procedures. This experiment was conducted at the Cinvestav-IPN facilities. The experimental schematic is illustrated in Fig. 6A and B. The implantation rate was obtained with the following formula: Table 1 provides a summary of the details for each experiment conducted at the respective centers. Table 1. | Experiment number | Study/exposure groups | Site of experiment | Number of animals used | Measured endpoints | |---|---|---|---|---| | Experiment 1 | Control, DEHP 20, 200, and 2,000 (µg/kg/day) | Cinvestav-IPN | 5 mice per group | Effects the development and quality of the blastocyst’s ICM and TE. | | Experiment 2 | Control, DEHP 20, 200, and 2,000 (µg/kg/day) | University of Illinois | 10 to 12 mice per group | Total number of recovered embryos Number of embryos at developmental stages Fragmentation rate and cell arrest rate | | Experiment 3 | Control, DEHP 20, 200, and 2,000 (µg/kg/day) | Cinvestav-IPN | 6 mice per group | Effect of DEHP on embryo Development and Implantation. | | Experiment 3A. DEHP-treated donors to untreated recipients | |||| | Experiment 3B. Untreated donors to DEHP-treated recipients | Statistical analysis Statistical analysis was performed using GraphPad Prism software (version 9.5.1, San Diego, CA, United States). Results are presented as mean±SEM. Data normality was assessed using the Shapiro–Wilk test. Outlier removal was performed with the (ROUT) method. For parametric data, 1-way ANOVA and Dunnett’s post hoc test were performed and for nonparametric data, 1-way ANOVA, Kruskall–Wallis, and Dunn’s post hoc test were used. Pearson correlation analysis was performed to assess the dose–response relationship (P 0.9 and a strong negative correlation if r<−0.9. Values of r = 1.000 indicated a linear dose–response pattern.

Experiment 1 Effect of DEHP on blastocyst development The results of the embryo quantification conducted at Cinvestav-IPN are documented in the study by Parra-Forero et al. (2019). Blastocyst development relies on morphological changes. To evaluate the effect of DEHP on morphology, we quantified early, late, and expanded blastocysts. The 200 and 2,000 μg/kg/day DEHP treatment groups had significantly higher percentages of early blastocysts compared with the control group and the 20 μg/kg/day DEHP group. Similarly, the 2,000 μg/kg/day DEHP group had a significantly decreased percentage of late blastocysts compared with the control group. However, all DEHP-exposed groups had similar numbers of expanded blastocysts to the control group (Fig. 1B). Effect of DEHP on ICM and TE quality The quality of the ICM was adversely affected in the group exposed to 2,000 μg/kg/day of DEHP. As illustrated in Fig. 1C, the percentage of blastocysts with good quality decreased by 21%, whereas the percentage of blastocysts with poor quality increased correspondingly. This difference was statistically significant when compared with the group exposed to 20 μg/kg/day. Exposure to 2,000 μg/kg/day of DEHP significantly decreased the percentage of blastocysts with good TE quality compared with control (Fig. 1D). The percentage of blastocysts with poor quality increased similarly in the groups exposed to 200 and 2,000 μg/kg/day. Experiment 2 Evaluation of the number and stage of embryo development We investigated the effects of DEHP on embryo formation and development under a subchronic exposure regimen, utilizing the experimental scheme illustrated in Fig. 1A. The number of embryos recovered from the perfused uterus and oviduct of mice exposed for 1 mo to environmentally relevant doses of 0, 20, 200, and 2,000 μg/kg/day DEHP was quantified. Subchronic exposure to 2,000 μg/kg/day DEHP resulted in a significant decrease in the total number of embryos recovered, ∼41.38% less compared with the control group. Embryonic development was also adversely affected in this group. The number of blastocysts was lower in the DEHP-treated groups compared with the control, whereas there was an increase in presumably single-cell embryos with developmental delay. This result was significantly different from the group exposed to DEHP 20 μg/kg/day. Data are presented as mean±SEM (Fig. 2A–E). Effect of DEHP on embryo fragmentation and cell arrest During embryo retrieval, the presence of presumptive embryos with different fragmentation patterns of the blastomeres was evident. In some cases, it was even impossible to define the developmental stage. The fragmentation rate and cell arrest rate were significantly higher in the group exposed to DEHP 2,000 μg/kg/day, with a 14.2% higher fragmentation rate and 15.6% higher cell arrest rate compared with the control group (see histogram in Fig. 2F and G). Figure 3A illustrates the normal and fragmentation patterns of embryo identification and classification. The populations of the presumptive embryos showed different developmental stages when recovery was performed at the time of blastocyst retrieval. Figure 3B shows representative images of the embryo populations obtained after flushing the uterus and oviducts of the control and DEHP 2,000 μg/kg/day treated groups. Effect of DEHP on GATA6, OCT4, and CDX2 in blastocysts The transcription factors GATA6 and OCT4 are evolutionarily conserved in both mice and humans (Frum et al. 2013; Schrode et al. 2014; Rossant and Tam 2022). The co-expression of these molecules plays a highly specialized role in the formation of the ICM, whereas CDX2 is crucial for regulating TE formation (Karasek et al. 2020). Given that DEHP affects the morphology of both ICM and TE, we evaluated the spatiotemporal expression of factors that are determinants for pluripotency activation, inhibition of differentiation within the ICM lineage, and TE cell functionalization. Figure 4A presents representative images of blastocysts immunostained for GATA6, OCT4, and CDX2 across the DEHP exposure groups. The patterns of GATA6 and OCT4 in the control group and the DEHP 20 μg/kg/day group were similar, with more specific localization in the blastocysts of the DEHP 20 μg/kg/day group. The patterns of GATA6 and OCT4 varied according to the developmental stage, as different fluorescence levels were observed in the ICM and TE cells of the control group blastocysts. In the DEHP 20 μg/kg/day group, a site-specific co-expression of GATA6 and OCT4 was observed, consistent with other reports, indicating compatibility with ICM formation. As a control, and to corroborate the co-expression of the transcription factors GATA6 (green) and OCT4 (red) in the ICM, the intersection of the 2 signals was observed, resulting in orange signals. A representative image is shown in Fig. 4B. Representative blastocysts from the DEHP 200 and 2,000 μg/kg/day groups exhibited alterations in blastocoel formation and the definition of the ICM and TE (Fig. 4A). Although the embryos appeared to be at the same developmental stage, the pattern and fluorescence intensity of GATA6, OCT4, and CDX2 varied between groups, with lower and more diffuse expression observed in the DEHP 2,000 μg/kg/day group. Figure 4B shows a representative picture of GATA6 and OCT4 co-expression in ICM. The histograms in Fig. 4C–E illustrate the expression levels of GATA6, OCT4, and CDX2 in the control group and those treated with DEHP (20 to 2,000 μg/kg/day). Effect of DEHP on the expression pattern of GATA6, OCT4-Positive cells in the ICM, and CDX2-Positive cells in the TE We quantified the positive cells for each transcription factor to investigate the expression of GATA6 and OCT4 within the ICM and CDX2 in the TE. Our analysis revealed distinct patterns of intensity and anatomical organization among the cells in the ICM, as illustrated in Fig. 5A, the histograms indicate significant reductions in the levels of GATA6 and OCT4 in the DEHP 2,000 μg/kg/day group compared with control. Additionally, CDX2 expression was reduced in both the DEHP 200 and 2,000 μg/kg/day groups compared with control (see Fig. 5B–D). Experiment 3 Effect of DEHP on embryo implantation rate The blastocyst quality assessment and the evaluation of transcription factors indicated that DEHP 200 and 2,000 μg/kg/day compromise blastocyst structure and morphology and potentially its functionality. To quantify the functional consequences of these effects, we evaluated the implantation capacity of DEHP-exposed blastocysts by using both DEHP-exposed and unexposed uteri (Fig. 6A and B). The results demonstrated significant differences in implantation rates compared with the control group at the 2,000 μg/kg/day dose in both experiments, whereas the 200 μg/kg/day dose showed differences only in implantation experiment 3B (Fig. 6C and D). Importantly, the results highlighted that DEHP exposure of donor embryos as well as DEHP exposure of recipient dams led to decreased implantation rates. Figure 6E presents representative images of the implantation patterns for the 2 treatment schemes. The uteri, stained with Chicago blue, exhibited variable patterns, as shown in the images. The implantation sites were confirmed as implanted embryos. These findings suggest that DEHP affects not only the blastocyst but also the uterine environment, as evidenced by the decreased implantation rates and the differences in Chicago blue staining patterns of uteri from female mice (recipient) treated with DEHP at 200 and 2,000 μg/kg/day. The results of several variables showed an apparent dose–response behavior. To verify this, we conducted Pearson’s correlation analysis. Table 2 summarizes the tendency toward linearity of the variables in each group. For the embryo recovery data, we observed a strong positive correlation between the variables Number of embryos and Number of blastocysts in the control group (r = 0.9544), DEHP 20 (r = 0.9513), and DEHP 200 (r = 0.9612). However, in the DEHP 2000 group, no strong positive or negative correlations were detected. Regarding the blastocyst quality and development analysis data, we identified notable negative and positive correlations in both the control group and the DEHP-exposed groups (20 to 2,000 μg/kg/day). Interestingly, the DEHP 2,000 μg/kg/day group exhibited extreme positive relationships in the following variables: Early blastocyst and Poor TE quality (r = 1.000), and Late blastocyst and Good ICM quality (r = 1.000). Additionally, the variables involved in the evaluation of GATA6, OCT4, and CDX2 expression showed strong positive correlations with CDX2 expression in the DEHP 2,000 μg/kg/day group. Table 2. | Experiment 1 | |||||| |---|---|---|---|---|---|---| | CONTROL | |||||| | Variables | r (Pearson correlation coefficient) | P-value | r (Pearson correlation coefficient) | P-value | r (Pearson correlation coefficient) | P-value | | Early blastocyst | Poor ICM quality | Fair TE quality | Poor TE quality | ||| | 0.9784876 | 0.003775 | −0.903551 | 0.035432 | 0.9783225 | 0.003819 | | | Late blastocyst | Good TE quality | ||||| | −0.90148409 | 0.036565 | ||||| | Poor ICM quality | Fair TE quality | Poor TE quality | |||| | −0.92390208 | 0.02491 | 0.99601993 | 0.000301 | ||| | Good TM quality | Fair TE quality | ||||| | −0.97840533 | 0.003797 | ||||| | Fair TE quality | Poor TE quality | ||||| | −0.9021441 | 0.036202 | ||||| | DEHP 20 μg/kg/day | |||||| | Early blastocyst | Good ICM quality | Poor ICM quality | Poor TE quality | ||| | −0.91415 | 0.029804 | 0.9627151 | 0.008594 | 1 | 0 | | | Late blastocyst | Good ICM quality | Fair ICM quality | |||| | 0.96403138 | 0.008144 | −0.97080477 | 0.005962 | ||| | Good ICM quality | Fair ICM quality | Poor ICM quality | Poor TE quality | ||| | −0.96026904 | 0.00945 | −0.95711243 | 0.010593 | −0.91414988 | 0.029804 | | | Poor ICM quality | Fair ICM quality | Poor ICM quality | |||| | −0.92791527 | 0.02298 | 0.96271508 | 0.008594 | ||| | DEHP 200 μg/kg/day | |||||| | Early blastocyst | Good TE quality | Fair TE quality | Poor TE quality | ||| | −0.925984 | 0.023902 | 0.9012698 | 0.036684 | 1 | 0 | | | Late blastocyst | Expanded blastocyst | Fair ICM quality | |||| | −0.99251729 | 0.000776 | −0.9416423 | 0.016774 | ||| | Expanded blastocyst | Fair ICM quality | ||||| | 0.93013936 | 0.021932 | ||||| | Good ICM quality | Fair ICM quality | ||||| | −0.90198655 | 0.036289 | ||||| | Good TM quality | Fair TE quality | Poor TE quality | |||| | −0.99814361 | 9.60E-05 | −0.92598397 | 0.023902 | ||| | Fair TE quality | Fair TE quality | ||||| | 0.9012698 | 0.036684 | ||||| | DEHP 2,000 μg/kg/day | |||||| | Early blastocyst | Poor TE quality | ||||| | 1 | 0 | ||||| | Late blastocyst | Good ICM quality | ||||| | 1 | 0 | ||||| | Experiment 2 | |||||| | CONTROL | |||||| | Number embryos | Number blastocyst | ||||| | 0.954396854 | 6.38E-05 | ||||| | DEHP 20 μg/kg/day | |||||| | Number embryos | Number blastocyst | ||||| | 0.951285222 | 2.32E-05 | ||||| | DEHP 200 μg/kg/day | |||||| | Number embryos | Number blastocyst | ||||| | 0.961223296 | 3.64E-05 | ||||| | DEHP 2,000 μg/kg/day | |||||| | AUF GATA6 | OCT4 positive cells | ||||| | 0.98727702 | 0.001719 | ||||| | AUF CDX2 | CDX2 positive cells | ||||| | −0.914308 | 0.029722 | “E” in scientific notation stands for “exponent” or “power of 10.”

The study of the impacts of DEHP exposure on reproduction is crucial, as DEHP is one of the most widely used plasticizers. Its extensive application in various products leads to accumulation in numerous ecological sources, making it ubiquitous (Qu et al. 2022). Low-dose exposures can produce significant molecular effects, potentially altering systemic and reproductive functions (Yu et al. 2020; Lee et al. 2021). Consequently, environmentally relevant scenarios vary greatly, with occupational exposure being of particular concern (Fréry et al. 2020). Furthermore, the increase in plasticizer production has been accompanied by a global decline in human fertility (Pogrmic-Majkic et al. 2022; Nobles et al. 2023). Various studies have demonstrated that the biological activity of DEHP can adversely affect the reproductive system (Chiang et al. 2020; Pérez et al. 2020) and in vivo studies have shown that DEHP reduces the number and quality of mouse embryos, but the mechanism remains unclear (Parra-Forero et al. 2019; Arcanjo et al. 2023). Our study demonstrates that exposure to environmentally relevant doses of DEHP affects preimplantation embryos by decreasing the numbers of embryos that develop to blastocysts, and reducing the expression of transcription factors (GATA6, OCT4, and CDX2), which are crucial for embryonic development and implantation. We further show that DEHP exposure of embryos as well as the uterus leads to reduced implantation rates in embryo transfer studies. The effect of DEHP has been associated with alterations in the hypothalamic–pituitary–ovarian (HPO) axis (Martínez-Ibarra et al. 2024). In our results from daily estrous cycle monitoring, we found no significant differences between the DEHP-exposed groups (Fig. S4). These findings are consistent with those reported by Laws et al. (2023), who conducted a 1-month exposure study with DEHP doses of 0.15 ppm, 1.5 ppm, and 1,500 ppm in a feed formulation. Their study indicated that the effect on estrous cyclicity could be influenced by an increase in the number of days in estrus after 3 and 5 months of exposure. The potential impact on the HPO axis cannot yet be ruled out. A Bayesian benchmark dose modeling analysis, focusing on endpoints such as hormonal levels and ovarian follicle counts, revealed increased sensitivity to serum progesterone in 10- and 30-day oral administration studies (Vieira Silva et al. 2025). Alterations in systemic progesterone levels are likely to affect pregnancy (Milligan and Finn 1997), particularly in the early stages (Shehata et al. 2023). Future studies should investigate the influence of hormonal variations on different reproductive endpoints, with a focus on progesterone, which appears to be highly sensitive to variations in DEHP and/or its metabolites. Embryonic development relies on a proper oviductal environment and correct embryonic cleavage (Kölle et al. 2020; Leung et al. 2023; Nabeel and Nowak 2024). At least 8 DEHP metabolites have been detected in human follicular fluid (Bellavia et al. 2023; Gokyer et al. 2025), as well as in serum, amniotic fluid, ovary, and oviduct in various species (Katsikantami et al. 2020; Li et al. 2020). Furthermore, they have been found in urine of women with fertility problems attending assisted reproduction clinics (Machtinger et al. 2018; Deng et al. 2020). Detection of DEHP metabolites in urine was associated with a decreased rate of high-quality D3 and D5 embryos (Deng et al. 2020; Tian et al. 2024). Our data show a decrease in the number of blastocysts recovered from DEHP-exposed mice compared with controls. These findings are in agreement with an in vitro study of embryos exposed to mono(2-ethylhexyl) phthalate (MEHP), in which blastocyst hatching was reduced at doses of 100 and 1,000 µM (Arcanjo et al. 2023). It is plausible that DEHP was metabolized in our model, and these metabolites also exert a detrimental effect on blastocyst development, as evidenced by the presence of embryos at earlier developmental stages (1-cell to morula stage) and fragmented embryos. Embryo fragmentation is often reported, but the causes remain unclear (Cecchele et al. 2022). Several mechanisms have been associated with this phenomenon, such as multinucleation in blastocyst cells, depletion of organelles, and essential proteins (Antczak and Van Blerkom 1999; Coticchio et al. 2021). These events can potentially alter structural conformation and affect fate decision processes in embryonic development (Alikani 2005; Budrewicz and Chavez 2024). Our results demonstrate an increase in fragmentation rate and cell arrest rate induced by DEHP. These effects were significantly more pronounced in the group exposed to DEHP at 2,000 μg/kg/day, with a 14.2% higher fragmentation rate and 15.6% higher cell arrest rate compared with the control group. This phenomenon has been reported by others for DEHP (Schmidt et al. 2012) and MEHP (Grossman et al. 2012; Arcanjo et al. 2023). Likely, due to the multifactorial nature of systemic events in our in vivo study, these occurrences also result from the formation of secondary metabolites that simultaneously converge on the embryos within the oviduct. Our experimental design allowed for the recovery of embryos at the appropriate time to obtain the majority of embryos at the blastocyst stage, assuming that the appearance of embryos at earlier stages would indicate cell division arrest (Fenelon 2024). In our control group, ∼70% of the embryos recovered were blastocysts, whereas the group exposed to 2,000 μg/kg/day DEHP had ∼50% blastocyst formation. Consecutive cell divisions constitute the cleavage of the embryo until cavitation occurs, polarizing the embryo and morphologically separating the ICM from the TE, allowing one to perform classification of the developmental stage into early, late, and expanded blastocyst (Zhu and Zernicka-Goetz 2020). Our data show an increase in the percentage of early blastocysts with DEHP doses of 200 and 2,000 μg/kg/day compared with the control, with a decrease in late blastocysts observed at the highest DEHP dose. Parra-Forero et al. 2019 observed a reduction in the number of embryos collected in the group exposed to DEHP at a dosage of 2,000 μg/kg/day (7%), whereas at the University of Illinois, the reduction was 18%. Regarding the decrease in the number of recovered blastocysts, Cinvestav-IPN reported significant differences between the DEHP 200 μg/kg/day (27%) and 2,000 μg/kg/day (34.4%) groups. At the University of Illinois, the DEHP 200 μg/kg/day group exhibited a 28% decrease, and the DEHP 2,000 μg/kg/day group showed a 37.2% reduction in blastocysts compared with the control group. The cell arrest rate was elevated at both centers. At Cinvestav-IPN, the DEHP 200 and 2,000 μg/kg/day groups showed arrest at the 1-cell stage (3.7% and 10.6%), 2-cell stage (7.65% and 24.2%), and morula stage (7.9% and 27.75%). The University of Illinois reported an increase in 1-cell arrest (6.97%) in the group exposed to DEHP 2,000 μg/kg/day. These results are consistent with an in vitro MEHP exposure study, which reported a reduced number of blastocysts formed (Arcanjo et al. 2023). Additionally, MEHP exposure has been associated with increased concentrations of reactive oxygen species and alterations in the expression of genes involved in metabolic processes (Kalo et al. 2019; Roth et al. 2020). Although MEHP is one of the most studied metabolites, the variability of reported effects suggests a simultaneous biological impact of DEHP and its metabolites, adding complexity to in vivo studies and the determination of their mechanisms of action. The specific configuration of the ICM is considered a marker of implantation potential and embryo viability (Balaban and Gardner 2013; Irani et al. 2017; Ai et al. 2021; Sivanantham et al. 2022). Although the morphological evaluation of embryos is subjective, a study evaluating 3,786 human blastocysts determined that ICM quality is the best predictor of blastocyst viability (Sivanantham et al. 2022). To our knowledge, no previous literature evaluated the quality of blastocysts under subchronic exposure to environmentally relevant doses of DEHP. In an in vivo experiment with C3H/N mice exposed to DEHP (0 to 500 mg/kg) for 8 wk, no significant differences were found in the number of degenerated blastocysts (Schmidt et al. 2012). In another study, DEHP (0 to 5 mg/kg/day) was used from the first day of gestation until the end of lactation in mice, and negative effects were found in the regulation of genes involved in the formation of the ICM and TE (Pocar et al. 2017). The correlations obtained in this study reveal different patterns for each dose. In the Control group and the DEHP 20 μg/kg/day group, the significance of high-quality ICM and its regulatory role in blastocyst development was emphasized. Expected biological outcomes, such as the positive relationship between early blastocyst formation and the poor quality of the ICM and TE, were observed, indicating that the establishment of ICM and TE begins in the early embryo, and is understood as a transitional period (Fischer et al. 2020; Guo et al. 2025). The transcription factors OCT4 and GATA6 are evolutionarily conserved in mice and humans and the co-expression of these molecules plays a highly specialized role in the formation of the ICM, meanwhile, CDX2 regulates the formation of the TE (Rossant and Tam 2022). According to our results, DEHP affects the morphology of both the ICM and TE. To understand the underlying mechanism, we evaluated the expression of these important factors that determine the activation of pluripotency and the inhibition of differentiation in the ICM lineage (Karasek et al. 2020). OCT4 expression decreased in intensity as the DEHP concentration increased. Additionally, DEHP exposure at 200 and 2,000 μg/kg/day decreased CDX2 expression compared with the control group. In an in vitro study with bovine oocytes exposed to MEHP, alterations in the gene expression of POU5F1 (OCT4) were observed at a concentration of 20 nM (Grossman et al. 2012). Pocar et al. (2017) observed in a transgenerational in vivo study that DEHP tended to increase the expression of OCT4 in the F2 and F3 generations after administering 0.05 and 5 mg/kg/day of DEHP. GATA6 and CDX2 expression have been evaluated and documented in mouse embryos exposed to various toxicants (Liu et al. 2022; Zhang et al. 2022, 2023). A recent study found that the Nrf2-Notch1 and Nf2 signaling pathways regulate the decrease in OCT4 expression accompanied by ectopic CDX2 expression in blastocysts (Li et al. 2024b). These data suggest that at a dose of 2,000 μg/kg/day, DEHP affects the expression of GATA6 and CDX2. Further studies are required to elucidate the mechanisms and implications of the coexpression of OCT4, GATA6, and CDX2 during the transition from early to late blastocyst stages in the formation of the ICM and TE. Our results indicate that DEHP exposure leads to a decrease in the expression of pluripotency markers in blastocysts, likely impairing the functional cell specialization required for the formation of ICM and TE, which are essential for the implantation process (Rosner and Hengstschläger 2024). In this study, we evaluated the implantation rate by transcervical transfer of expanded blastocysts to assess their functionality based on implantation capacity. In Experiment 3A, blastocysts from the 2,000 μg/kg/day group exhibited a reduced ability to implant in the uteri of animals exposed to the vehicle (corn oil). In Experiment 3B, we investigated the uterine-level effects of DEHP by transferring embryos from untreated animals to DEHP-treated animals; the 200 and 2,000 μg/kg/day doses showed a higher percentage of implantation failure, indicating a possible dose-dependent uterine response. The results observed in this study support an effect of DEHP on the uterus. Multiple variables may contribute to the decreased embryo implantation rate caused by phthalate exposure (Basso et al. 2022). In a study by Li et al. (2012), pregnant mice received DEHP at 0, 250, 500, and 1,000 mg/kg/day exhibited a decreased number of implantation sites and were associated with upregulation of genes encoding E-cadherin, estrogen receptor, and progesterone receptors, demonstrating that the HPO axis may be compromised by DEHP exposure. The vascular structure and uterine permeability are likely affected in these groups, because uterine vascular alterations have been reported for in vivo DEHP exposure studies. In 2018, Richardson et al. reported that DEHP at 200 μg/kg/day for 30 days in a mouse (CD-1) model caused reduced proliferation, accompanied by blood vessel dilation in the endometrium. Another transgenerational study of exposure during pregnancy with a mixture of phthalates, including DEHP, found similar results at a dose of 200 mg/kg/day (Li et al. 2020). Some of these changes can become pathological and have been associated with DEHP exposure, primarily due to the presence of DEHP and its metabolites in urine of affected patients (Koch and Angerer 2007). These include decidualization defects (Long et al. 2023), endometriosis (Wieczorek et al. 2022), risk of uterine leiomyoma (Iizuka et al. 2022), miscarriage (Kim et al. 2017), and recurrent implantation failure (Chang et al. 2021). In vitro studies in different uterine cell lines have demonstrated that treatment with DEHP or its metabolites can induce structural and functional changes (Kim et al. 2022; Lavogina et al. 2024). Our studies did not directly evaluate the effect of DEHP on the endometrium; however, they provide a foundation for specific uterus-focused studies to understand the reproductive pathophysiology of DEHP. The results of our studies reveal the high complexity of the response to DEHP exposure across various variables involved in embryonic development and subsequent implantation. A limitation of our study is the lack of hormonal evaluation, which would have been valuable for assessing the effects at the HPO axis level. However, the superovulation protocol was necessary to maximize the number of embryos per animal, thereby supporting the initiative to minimize the number of animals used, following the 3R principle. Consequently, hormonal levels likely responded to the exogenous stimulus rather than DEHP exposure. Furthermore, this study demonstrated the susceptibility induced by DEHP, leading to a decrease and spatial disruption in the expression of GATA6, OCT4, and particularly CDX2, which is crucial for TE formation and early pregnancy establishment. These findings, along with the results of the implantation assessment, provide a foundation for future studies on early implantation and elucidating the complex mechanisms underlying the response to DEHP exposure.

The results of this study demonstrate the deleterious effects of DEHP on blastocyst development and provide an informative basis for further investigation of DEHP at environmentally relevant doses. These data describe the complexity of the DEHP interaction and its linear behavior for some variables. Collectively, our findings suggest that DEHP and its metabolites, when distributed systemically, contribute to the structural alteration of embryos, as evidenced by the observed fragmentation and molecular effects on the expression of GATA6, OCT4, and CDX2. These effects may contribute to implantation failure (see Fig. 7). Supplementary Material Acknowledgments Thanks to Rachel Braz Arcanjo, Nastasia Lai, Tauseef Shah, Fernanda Avila and Arturo Sanchez for their help in tissue collection. Special thanks to the Flaws lab for their assistance in preparing the DEHP doses. We also appreciate the Core Facilities at the Carl R. Woese Institute for Genomic Biology for their technical assistance with image acquisition. Contributor Information Lyda Yuliana Parra-Forero, Department of Animal Sciences, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States; Department of Toxicology, Center for Research and Advanced Studies (Cinvestav-IPN), Ciudad de México 07360, Mexico. Isabel Hernández-Ochoa, Department of Toxicology, Center for Research and Advanced Studies (Cinvestav-IPN), Ciudad de México 07360, Mexico. Jodi A Flaws, Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, IL 61802, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States. Romana A Nowak, Department of Animal Sciences, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, United States. Author contributions Lyda Parra: Conceptualization, Visualization, Methodology, Writing—original draft, Investigation, Writing—Review & Editing. Isabel Hernandez: Conceptualization, Methodology, Formal analysis, Funding acquisition, Supervision, Investigation. Jodi Flaws: Conceptualization, Methodology, Resources, Methodology, Funding acquisition, Investigation. Romana Nowak: Conceptualization, Methodology, Writing—review & editing, Supervision, Funding acquisition, Investigation, Writing—Review & Editing. Supplementary material Supplementary material is available at Toxicological Sciences online. Funding This work was supported by a scholarship for graduate students from the National Council of Science and Technology-Mexico (CONACyT-Mexico) (Fellowship No. 426444) and the Grant National Institutes of Health (R21ES026388 and R01ES034113). Conflicts of interest 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. Data availability The raw data will be available upon reasonable request to the senior author.

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Supplementary Materials Data Availability Statement The raw data will be available upon reasonable request to the senior author.