Cardiac function depends on continuous oxidative metabolism, rendering cardiomyocytes highly vulnerable to oxygen deprivation. Here, we performed a genome-wide CRISPR interference (CRISPRi) screen in human iPSC-derived cardiomyocytes to identify genes that modulate survival during chronic hypoxia. This screen revealed that knockdown of basigin (BSG), a chaperone for the monocarboxylate transporters MCT1 and MCT4, confers robust protection. Canonically, hypoxic cells suppress pyruvate dehydrogenase (PDH) activity to reduce the oxidation of major fuel sources, thereby limiting TCA cycle flux, lowering oxygen consumption, and minimizing reactive oxygen species generated by an overly reduced electron transport chain (ETC). In contrast, we found that BSG inhibition reverses this response, prioritizing ATP maintenance during hypoxia and enhancing cardiomyocyte survival. Mechanistically, BSG loss restricts lactate efflux, leading to decreased PDH phosphorylation and increased glucose uptake for oxidation. Consistent with this, ETC subunits are more essential under hypoxia, highlighting cardiomyocytes' unusual reliance on aerobic ATP production even when oxygen is limited. These findings challenge prevailing models of hypoxic adaptation by revealing cardiomyocyte-specific bioenergetic requirements and motivating future therapeutic efforts.

Phthalates are associated with several reproductive disorders in women and reduce fertility in mice. They are also known to impair hepatic glycogen metabolism. Glucose is a crucial nutrient for the uterus, and glycogen buffers glucose concentration in the endometrium. The objective of this study was to investigate how long-term exposure to di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP) alters glycogen metabolism in the murine endometrium. Six-week-old female mice were fed chow containing vehicle or DEHP or DiNP at 0.15, 1.5, and 1500 parts per million (ppm) ad libitum for 9 months. Uteri were collected at diestrus. DEHP significantly reduced glycogen levels in the glandular epithelium (GE) and luminal epithelium (LE). In the stroma, both 1.5 and 1500 ppm groups had significantly lower glycogen. In the DiNP-treated mice, all three concentrations significantly decreased glycogen in GE, LE, and stroma. Neither phthalate altered mRNA levels of hexokinase1 (Hk1), glycogen synthase 1 (Gys1), glycogen phosphorylase M (Pygm), or glucose-6-phosphatase 3 (G6pc3). Immunohistochemistry showed that both phthalates increased HK1 levels in the stroma but not the epithelium. DEHP and DiNP (1500 ppm) increased PYGM in GE, LE, and stroma. DiNP (1500 ppm) significantly lowered G6PC3 in LE compared to all other groups. In the GE, both 1.5 and 1500 ppm DiNP decreased the immunostaining of G6PC3 compared to control and 0.15 ppm DiNP. Our results show that phthalates alter endometrial glycogen levels and expression of key enzymes. These findings are consistent with altered glycogen metabolism, which could alter endometrial glucose metabolism.

Abstract:Extracellular vesicles (EVs) are membrane-bound particles that play an important role in cell-to-cell communication. Embryo-maternal communications are vital for early embryo development and implantation. Uterus-derived EVs play a crucial role in embryo-maternal crosstalk. The current study investigated how progesterone regulates protein cargoes in EVs derived from bovine uterine epithelial cells (BUTE cell-EVs). After isolation, BUTE cell-EVs were characterized using scanning transmission electron microscopy, nanoparticle tracking analysis, western blotting, and mass spectrometry. BUTE cells were treated with 0 or 300 nM progesterone to investigate potential regulation of protein cargoes in BUTE cell-EVs. We determined that BUTE cell-EVs have the characteristic cup shape structure, ranging in size from 20 to 550 nm, and expressed CD9 and CD81, as well as other known proteins for EV biogenesis. The number of BUTE cell-EVs released was not affected by progesterone. Proteomic analysis showed that BUTE cell-EVs contained proteins with functions related to implantation, uterine receptivity, and trophoblast differentiation. Progesterone altered the EV protein cargo, with 45 proteins differentially expressed compared to control. Differentially expressed proteins related to implantation and adhesion included galectin-3 and galectin-3-binding protein. The actin-binding protein gelsolin was also found to be differentially expressed in progesterone-treated BUTE cell-EVs. We also showed that BUTE cell-EVs were successfully internalized into bovine embryos. In conclusion, the proteomic profile of BUTE cell-EVs was altered by progesterone, and these EVs are internalized into the bovine embryos, delivering proteins into bovine embryos that are important for implantation, embryo adhesion, and proper embryo metabolism and development. Lay summary:Communication between the womb (uterus) and the developing embryo is essential for successful pregnancy. Tiny particles called EVs are released from cells in the uterus and carry proteins and other molecules that help the embryo grow and attach to the uterus. In this study, we investigated how the hormone progesterone influences the contents of EVs released from the cells (epithelial cells) that line the cow's uterus. We found that progesterone alters the amount of specific proteins in these vesicles without affecting the number of vesicles released. Many of the altered proteins are involved in preparing the uterus for implantation and supporting embryo development. We also demonstrated that these vesicles can be taken up by early-stage cow embryos. Our findings suggest that progesterone changes proteins that are packed in these vesicles, helping create a receptive environment for implantation and early development.

Phthalates are a class of synthetic compounds, known as endocrine-disrupting chemicals, widely used as plasticizers in consumer products, including personal care items, medical devices, and food packaging. Two common phthalates, di(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DiNP), have been associated with adverse effects on female reproductive health. This study investigated the effects of acute DEHP and DiNP exposure on uterine inflammation and oxidative stress in adult female CD-1 mice. Mice were orally dosed for 10 days with vehicle control, DEHP (20 μg/kg/day, 200 μg/kg/day, or 200 mg/kg/day), or DiNP (20 μg/kg/day, 100 μg/kg/day, or 200 mg/kg/day). Uteri were collected during diestrus for histological and gene expression analyses. Quantitative PCR (qPCR) showed that DEHP (20 and 200 μg/kg/day) and DiNP (200 mg/kg/day) increased expression of inflammasome-related genes (Il18, Il1β, and Nlrp3). DiNP at 200 mg/kg/day also increased Il10 expression. Oxidative stress genes revealed DEHP increased Prdx2 expression at all doses without affecting Sod1, Cat, or Gpx1. However, DiNP increased Prdx2 at 20 μg/kg/day but reduced Sod1, Cat, and Gpx1 at higher doses. Histological analysis revealed that high-dose DiNP reduced outer myometrium thickness and luminal epithelial cell height, while DEHP only affected the cell height at the highest dose. Macrophage and other mononuclear phagocytic cell infiltration increased with DEHP (20 and 200 μg/kg/day) and all doses of DiNP. while cell proliferation was only changed in DEHP (200 μg/kg/day). Together, these findings demonstrate that acute exposure to DEHP and DiNP induces uterine inflammatory and oxidative stress responses, with distinct dose-dependent effects for each phthalate.

Because the blastocyst is a critical stage of early embryonic development, developmental abnormalities can serve as sensitive biomarkers of reproductive toxicity. This review focuses on the impact of various endocrine disrupting chemicals (EDCs) including phthalates, bisphenols, parabens and per- and polyfluoroalkyl substances on blastocyst formation, structure, and function. Murine blastocyst development models offer a controlled and reproducible platform to study cell differentiation, implantation potential, and epigenetic regulation. In vitro systems allow simulation of internal EDC concentrations found in human biological fluids, enabling dose-response relationships that reflect real world exposure. EDCs present in reproductive fluids such as follicular, oviductal, and endometrial fluid can impair oocyte quality and compromise embryonic development, even in morphologically normal embryos. Blastocysts exposed to EDCs during development show alterations in lineage specification markers (e.g., OCT4, SOX2, CDX2), cytoskeletal organization, and nuclear integrity, suggesting disruption of key developmental pathways. This review systematically reviews in vitro and in vivo evidence, along with proposed mechanisms of action, for each group of EDCs. Importantly, only studies reporting blastocyst formation rates were included to ensure relevance and comparability. The synthesis identifies common toxicity patterns, potential synergistic effects, and transgenerational consequences. The findings have direct implications for human fertility, particularly in the context of assisted reproductive technologies (ART), and highlight the need for improved regulatory frameworks and biomarker validation. Overall, the blastocyst emerges as a powerful tool for understanding and mitigating the reproductive risks associated with environmental chemical exposure.

Diisononyl phthalate (DiNP), a plasticizer increasingly replacing di(2-ethylhexyl) phthalate, is an endocrine-disrupting chemical linked to female reproductive harm. Ingestion is the most common route of DiNP exposure, making the gastrointestinal tract and gut microbiome a direct target for endocrine-disrupting chemical exposure. This study examined the effects of acute DiNP exposure either in the absence or presence of a gut microbiome on uterine development. Female C57Bl/6 germ-free (-microbiome) 40-day-old mice were orally dosed, over 3 days, with either sterile phosphate-buffered (n = 8) to remain germ-free (GF, -microbiome) or with colon contents (n = 10) to develop a gut-microbiome (+microbiome). This was followed by a 10-day period where half of the -microbiome and +microbiome mice were orally dosed with corn oil while half were orally dosed with 200 μg/kg/day DiNP. The control group were specific pathogen-free conventionally housed mice born with a microbiome. Mice were euthanized in diestrus at the end of the 10 days. Uteri were collected for histological analyses. Uterine development was significantly delayed in GF mice, regardless of later microbiome reintroduction or DiNP exposure. Key findings included reduced uterine diameter, stroma area, and gland number, and thinner myometrial layers. Endometrial stromal cell proliferation was also lower in GF mice. DiNP exposure alone showed no significant effects. Estradiol levels and ovarian follicle counts were similar across groups, but GF mice had fewer, smaller litters in fertility tests. The study highlights that the gut microbiome critically influences postnatal uterine development, with its absence leading to persistent structural deficits. DiNP, at the tested dose, did not exacerbate these effects.

The global decline in human fertility has become an increasing public health concern, marked by notable regional disparities and a growing reliance on assisted reproductive technologies (ART). Among the environmental contributors to reproductive dysfunction, phthalates, ubiquitous endocrine-disrupting chemicals, have been implicated in adverse reproductive outcomes. This study aimed to evaluate the effects of a biologically relevant phthalate mixture on preimplantation embryo development using an in vitro mouse model. Dosing proportions were based on phthalate concentrations measured in urine samples from pregnant participants in the Illinois Kids Development Study (I-KIDS), with the following composition: 35% diethyl phthalate (DEP), 21% di(2-ethylhexyl) phthalate (DEHP), 15% dibutyl phthalate (DBP), 15% diisononyl phthalate (DiNP), 8% diisobutyl phthalate (DiBP), and 5% benzyl butyl phthalate (BBzP). Embryos were exposed to this mixture at concentrations of 0.001, 0.01, 0.1, and 1 μg/mL, alongside control groups (culture medium only and 0.075% DMSO vehicle control), from the zygote to the hatched blastocyst stage. Exposure resulted in a significant reduction in developmental progression, with increased cytoplasmic fragmentation observed during the 2- to 8-cell transition in embryos treated with 0.1 and 1 μg/mL. Concentrations of 0.01, 0.1, and 1 μg/mL caused a marked decrease in E-cadherin expression at the 8-cell stage, and a significant increase in micronucleus formation was observed at the blastocyst stage after exposure to 0.1 and 1 μg/mL. These findings suggest that phthalate exposure disrupts critical processes in early embryogenesis, including cell adhesion, and nuclear integrity, potentially compromising embryo viability.

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.

Parabens are chemicals widely used in personal care products and food as antimicrobial preservatives. They exhibit potential estrogenic activity by binding to estrogen receptors 1 and 2, classifying them as endocrine-disrupting chemicals. Given the substantial daily exposure of women to parabens, it is crucial to investigate their effects on the female reproductive system. Previous studies in mouse models have shown that paraben exposure impacts ovarian development, resulting in an increase in cystic follicles and a decrease in corpora lutea. However, the effects of parabens on embryo development have not been extensively studied. This study aimed to determine the impact of propylparaben exposure on preimplantation embryo development in vitro. We tested the effects of 0 (0.075 % DMSO), 0.5 μg/mL, 5.0 μg/mL, 10 μg/mL, and 15 μg/mL propylparaben on rate of development of mouse zygotes to hatched blastocyst stage, quantified the number of inner cell mass (ICM) and trophectoderm (TE) cells in hatched blastocysts, and the distribution of cytoskeletal F-actin. The percentage of hatched blastocysts was significantly decreased at 0.5 μg/mL and 10 μg/mL compared to controls. Propylparaben treatment did not alter TE cell numbers. However, treatment with 0.5 or 15 μg/mL significantly decreased the number of ICM cells compared to controls. Additionally, the intensity of phalloidin fluorescence staining for F-actin was significantly reduced at 10 μg/mL and 15 μg/mL propylparaben. In summary, our findings show that propylparaben exposure disrupts ICM formation, impacts the cytoskeletal filamentous actin (F-actin) network, and alters the rate of hatched blastocyst development in preimplantation mouse embryos.

The release of basigin protein by human placental trophoblast cells via extracellular vesicle release is a regulated process. This study shows interactions of trophoblast cells with uterine cells during early placental development. Successful pregnancy relies on the regulated invasion of trophoblast cells into the maternal endometrium and subsequent remodeling of spiral arteries. Various factors are involved in regulating these processes, including matrix metalloproteinases (MMPs), cytokines such as interleukins (IL) and transforming growth factor β (TGFβ), and hypoxic conditions. Basigin (BSG), a glycosylated protein, plays an important role in MMP induction and inflammation. The role of BSG during early stages of placental development is not yet clear, nor is the process by which BSG is secreted by trophoblast cells. This study investigated the mechanism of BSG secretion from trophoblast cells in extracellular vesicles (EVs), and whether BSG release in EVs is a regulated process. RT-PCR was used to identify BSG isoforms 2, 3, and 4 in the trophoblast-like cell lines JAR, JEG-3, and HTR-8/SVneo. BSG protein expression was confirmed in trophoblast cell lines and conditioned medium by immunoblotting. We confirmed that BSG is released from HTR-8/SVneo cells via EVs. Treatment of HTR-8/SVneo cells with the protein kinase C activator PMA increased release of BSG-containing EVs, whereas the protein kinase C inhibitor Bis reduced release. Hypoxia/reoxygenation increased BSG protein in released EVs. IL-1β enhanced, while TGF-β1 reduced BSG in released EVs. This effect occurred at the post-transcriptional level, as the quantity of EVs released and levels of BSG mRNA expression in HTR-8/SVneo cells were not altered. Our findings support that BSG, released via EVs, may play an important role in facilitating interactions between trophoblast cells and uterine cells during early stages of placental development.

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as pivotal mediators of intercellular communication. Embryo implantation is a critical process in early pregnancy and requires communication between the embryo and maternal uterus. EVs are important in coordinating the communication between the embryo and maternal uterus. This review explores EV biogenesis, molecular composition, and functional roles during implantation. It emphasizes the dynamic role of EVs in modulating the maternal-embryo dialogue, which is critical for establishing a receptive endometrium and facilitating successful implantation. EVs secreted by the embryo and endometrial cells have been shown to carry a diverse cargo of proteins, lipids, and miRNAs, which collectively influence key physiological processes, including immune tolerance, endometrial receptivity, and trophoblast invasion. EVs can be potential candidates as non-invasive biomarkers to assess the quality of embryos and uterine receptivity to enhance reproductive success. By providing a comprehensive overview of the current understanding of EVs in implantation, this chapter aims to highlight the significance of EVs in reproductive biology and their potential applications in improving fertility rates.

The female reproductive system ages before any other physiological system, making it a sensitive indicator of aging. Early reproductive aging is associated with the early onset of infertility and an increased risk of several diseases. During aging, systemic and reproductive oxidative stress and inflammation levels increase through inflammasome activation, leading to ovarian follicle loss. Other markers of reproductive aging include increased fibrosis and shortening of telomeres in ovarian cells. The factors that accelerate reproductive aging are unclear, but likely involve exposure to endocrine-disrupting chemicals such as phthalates. Di(2-ethylhexyl) phthalate (DEHP) is a widely used phthalate and humans are exposed to it daily. Several studies show that DEHP induces reproductive toxicity by affecting estrous cyclicity, follicle numbers, and hormone levels. However, little is known about the mechanisms underlying DEHP-induced early onset of reproductive aging. Thus, this study tested the hypothesis that dietary exposure to DEHP induces early reproductive aging by affecting inflammation, fibrosis, and the expression of telomere regulators and antioxidant enzymes. Adult CD-1 female mice were exposed to vehicle (corn oil) or DEHP (0.5, 1.5, or 1500 ppm) via the chow for 6 months. Exposure to DEHP increased the expression of antioxidant enzymes and Caspase 3, increased expression of telomere-associated genes, and increased fibrosis levels in the ovary. In addition, DEHP exposure for 6 months altered ovarian and systemic inflammatory status. Collectively, our novel data suggest that 6-month dietary exposure to DEHP may accelerate reproductive aging by affecting several reproductive aging markers in female mice.

The interaction of infection during gestation and insults later in life influences the molecular mechanisms in the hypothalamus that participate in pain sensation. The response of the hypothalamic transcriptome varies between sexes and can also affect synapses and immune signals. The findings from this study assist in the identification of agonists or antagonists that can guide pretranslational studies to ameliorate the effects of gestational insults interacting with postnatal challenges on physiological or behavioral disorders.

DNA methylation is an epigenetic modification that can alter gene expression, and the incidence can vary across developmental stages, inflammatory conditions, and sexes. The effects of viral maternal viral infection and sex on the DNA methylation patterns were studied in the hypothalamus of a pig model of immune activation during development. DNA methylation at single-base resolution in regions of high CpG density was measured on 24 individual hypothalamus samples using reduced representation bisulfite sequencing. Differential over- and under-methylated sites were identified and annotated to proximal genes and corresponding biological processes. A total of 120 sites were differentially methylated (FDR-adjusted p-value < 0.05) between maternal infection or sex groups. Among the 66 sites differentially methylated between groups exposed to inflammatory signals and control, most sites were over-methylated in the challenged group and included sites in the promoter regions of genes SIRT3 and NRBP1. Among the 54 differentially methylated sites between females and males, most sites were over-methylated in females and included sites in the promoter region of genes TNC and EIF4G1. The analysis of the genes proximal to the differentially methylated sites suggested that biological processes potentially impacted include immune response, neuron migration and ensheathment, peptide signaling, adaptive thermogenesis, and tissue development. These results suggest that translational studies should consider that the prolonged effect of maternal infection during gestation may be enacted through epigenetic regulatory mechanisms that may differ between sexes.

Di(2-ethylhexyl) phthalate and diisononyl phthalate are widely used as plasticizers in polyvinyl chloride products. Short-term exposures to phthalates affect hormone levels, ovarian follicle populations, and ovarian gene expression. However, limited data exist regarding the effects of long-term exposure to phthalates on reproductive functions. Thus, this study tested the hypothesis that short-term and long-term exposure to di(2-ethylhexyl) phthalate or diisononyl phthalate disrupts follicle dynamics, ovarian and pituitary gene expression, and hormone levels in female mice. Adult CD-1 female mice were exposed to vehicle, di(2-ethylhexyl) phthalate, or diisononyl phthalate (0.15 ppm, 1.5 ppm, or 1500 ppm) via the chow for 1 or 6 months. Short-term exposure to di(2-ethylhexyl) phthalate (0.15 ppm) and diisononyl phthalate (1.5 ppm) decreased serum follicle-stimulating hormone levels compared to control. Long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate (1500 ppm) increased the percentage of primordial follicles and decreased the percentages of preantral and antral follicles compared to control. Both phthalates increased follicle-stimulating hormone levels (di(2-ethylhexyl) phthalate at 1500 ppm; diisononyl phthalate at 1.5 ppm) and decreased luteinizing hormone levels (di(2-ethylhexyl) phthalate at 0.15 and 1.5 ppm; diisononyl phthalate at 1.5 ppm and 1500 ppm) compared to control. Furthermore, both phthalates altered the expression of pituitary gonadotropin subunit genes (Cga, Fshb, and Lhb) and a transcription factor (Nr5a1) that regulates gonadotropin synthesis. These data indicate that long-term exposure to di(2-ethylhexyl) phthalate and diisononyl phthalate alters follicle growth dynamics in the ovary and the expression of gonadotropin subunit genes in the pituitary and consequently luteinizing hormone and follicle-stimulating hormone synthesis.

The combination of a good quality embryo and proper maternal health factors promise higher chances of a successful in vitro fertilization (IVF) procedure leading to clinical pregnancy and live birth. Of these two factors, selection of a good embryo is a controllable aspect. The current gold standard in clinical practice is visual assessment of an embryo based on its morphological appearance by trained embryologists. More recently, machine learning has been incorporated into embryo selection “packages”. Here, we report EVATOM: a machine-learning assisted embryo health assessment tool utilizing an optical quantitative phase imaging technique called artificial confocal microscopy (ACM). We present a label-free nucleus detection method with, to the best of our knowledge, novel quantitative embryo health biomarkers. Two viability assessment models are presented for grading embryos into two classes: healthy/intermediate (H/I) or sick (S) class. The models achieve a weighted F1 score of 1.0 and 0.99 respectively on the in-distribution test set of 72 fixed embryos and a weighted F1 score of 0.9 and 0.95 respectively on the out-of-distribution test dataset of 19 time-instances from 8 live embryos.

The data is an example dataset for mouse embryo viability assay presented in "EVATOM: a novel embryo health assessment tool." It contains three folders: healthy: a H/I class embryo, sick: a S class embryo and sparse_prediction: for demonstrating sparse prediction functionality on select z-slices of live embryo. Healthy and sick folders, each have two subfolders: 'cropped'-containing LS-GLIM quantitative phase images and 'new'-containing corresponding nucleus predictions from NPM. Sparse_prediction folder has select z-slices in 'overlapped' folder with raw file needed for IBM prediction 'unknown.csv' and IBM prediction example 'check.csv'.MATLAB app for demonstration of EVATOM is also provided.modelp99.pth is the trained IBMfinal_model.h5 is the trained NPMmodel_94202.mat is the trained FBMSupplementary movies are also provided.Github repository for model inference scripts is linked as related materials.