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.

Recently, we have shown that exposure to the ultraviolet filter benzophenone-3 (BP3) during secretory differentiation disrupts prolactin signaling pathway and milk protein expression in murine mammary organoids. The present study aimed to evaluate whether intrauterine exposure to BP3 modifies the functional differentiation of the lactating mammary gland in F1 female mice. To this end, pregnant mice (F0) were dermally exposed to BP3 (0.15 or 50 mg/kg/day) or the control vehicle (sesame oil), from gestation days 8.5-18.5. After delivery, F0 dams nursed their pups, and F1 female offspring were subsequently bred at 8 weeks of age. Lactation day (LD) 1 was defined as the day following the birth of F2 litters and lactating mammary glands of F1 females were examined on LD10. BP3 exposure did not affect the body weight of the F1 females or that of their F2 litters. Intrauterine exposure to BP3 significantly altered alveolar size and myoepithelial cell density at mid-lactation. However, the expression of milk proteins and the mRNA levels of Stat5a and Elf5 were similar to those of control animals. These findings reveal a subclinical mammary phenotype in which intrauterine BP3 exposure alters alveolar organization and the myoepithelial compartment while preserving lactational output, consistent with a reduction in mammary functional reserve without overt impairment of milk production or offspring growth.

Prednisone is commonly used to control autoimmune and inflammatory diseases during pregnancy, but its potential effects on fetal skeletal development remain incompletely characterized. This study investigated the dose- and exposure-period-related effects of prenatal prednisone exposure (PPE) on fetal long-bone development in mice and explored the cellular mechanism involving chondrocyte-to-osteoblast transdifferentiation. Pregnant mice received prednisone at 0.25 or 1.0 mg/kg/day throughout gestation or 1.0 mg/kg/day during defined gestational periods. Fetal femurs were collected on gestational day 18 (GD18) for histomorphometric, histochemical, and immunofluorescence analyses. PPE shortened fetal femurs, reduced the primary ossification center (POC), narrowed the proliferative zone (PZ), and expanded the hypertrophic zone (HZ), with generally greater changes after higher-dose or longer-duration exposure. Quantitative cellular analysis demonstrated fewer proliferative chondrocytes, accumulation of hypertrophic chondrocytes, and reduced osteoblast numbers within the POC. Consistently, PPE decreased SRY-box transcription factor 9 (SOX9) and runt-related transcription factor 2 (RUNX2) protein expression while increasing type X collagen (COL10) expression. The accumulation of hypertrophic chondrocytes, together with insufficient osteoblast formation, indicates that PPE impairs the cellular transition from hypertrophic chondrocytes to osteoblasts. Collectively, impaired chondrocyte-to-osteoblast transdifferentiation represents a cellular mechanism contributing to PPE-induced fetal long-bone dysplasia.

Chronic exposure to the widely used organophosphate pesticide chlorpyrifos has been associated with reproductive dysfunction; however, the early subclinical events preceding overt reproductive dysfunction remain poorly understood due to cell-specific and non-monotonic responses to the toxicant. The present study investigated the effect of chronic CPF exposure on the spermatogenic niche and sperm epigenome in a mouse model. Despite the absence of detectable oxidative stress or reproductive toxicity, chronic CPF exposure induced significant changes in the testicular microenvironment. Testicular proteomic analysis revealed dysregulated expression of proteins involved in extracellular matrix remodeling in CPF-exposed mice. These changes were associated with increased expression of Ctnnb1 and Axin1 indicating modulation of β-catenin-associated signaling in the testis. Furthermore, CPF exposure was associated with dysregulation of Sertoli cell function characterized by a significant reduction in Kitlg expression, increased c-kit levels, and reduced expression of the gap junction protein Connexin 43. CPF exposure also led to a significant dose-dependent increase in the expression of Tet1 suggesting altered epigenetic regulation in the testis. Interestingly, there was a trend toward increased sperm DNA methylation in CPF-exposed mice. Overall, these findings reveal that chronic CPF exposure induces early remodeling of the spermatogenic niche and alters testicular epigenetic regulation prior to overt reproductive dysfunction, identifying the Sertoli cell-extracellular matrix axis as a potential early target underlying CPF-induced testicular toxicity.

Plastic production has been increasing exponentially. Throughout their lifespan, plastics degrade into smaller particles that accumulate in our bodies and the environment. Recent studies found these plastic particles can cross the placental barrier and reach the fetus. However, the impact of plastic particles on placental function is still unknown. We hypothesized that nanoplastics would disrupt placental growth and function, specifically focusing on transforming growth factor beta (TGFβ) signaling. To understand the impact of plastic particles on the placenta, we orally exposed pregnant CD-1 mice to 50 nm or 200 nm polystyrene plastic particles from gestation day 8 to day 15 at a human-relevant concentration of 5 mg/kg/day. After euthanasia on day 15, placenta and fetus weights were recorded, and tissues were prepared for histomorphology and gene expression analysis. We observed a statistically significant decrease in the area of the decidua in the placentas for the 200 nm treatment group and a borderline significant decrease in decidua area for the 50 nm treatment group compared to control. However, when separated by sex, only the male decidua were significantly decreased in the 200 nm group. Gene expression analysis of key signaling factors in the TGFβ pathway identified increased expression of Smad2 and Smad3, which may be suppressing estrogen receptor signaling. Overall, both particle sizes disrupted placenta structure and signaling in a sex-dependent manner.

Whether PM2.5-induced spermatocyte injury engages the cGAS-STING innate immune pathway remains unknown. This study integrated network pharmacology, GEO transcriptomic profiling (GSE189187), gene set enrichment analysis, molecular docking with positive-control benchmarking, single-cell RNA sequencing, and in vitro validation in GC-2 spermatocyte cells to address this question. Among 35 overlapping candidates between 447 AS-IV putative targets and 102 cGAS-STING/inflammation genes, Hmox1 was the sole gene significantly upregulated by PM2.5 (log2FC = 2.15, AUC = 1.000). KEGG analysis identified six enriched innate immunity pathways, with the Cytosolic DNA-sensing pathway at adjusted P = 8.1 × 10[-6]. Molecular docking demonstrated that AS-IV bound cGAS (-10.2 kcal/mol) and STING (-11.3 kcal/mol) with affinities exceeding known inhibitors G150 (-8.8) and H-151 (-8.6). In GC-2 cells, PM2.5 (200 μg/mL) increased ROS 5.35-fold, elevated apoptosis to 42.83%, and activated cGAS-STING signaling. AS-IV co-treatment restored viability to 83.3%, enhanced proliferation to 37.96%, significantly suppressed cGAS/p-TBK1/p-IRF3, and activated Nrf2/HMOX1 (HMOX1 protein 1.99 ± 0.05-fold; mRNA 6.44-fold), reducing apoptosis to 18.27%, ROS, and LDH release, and normalizing the Bax/Bcl2 ratio from 5.0 to 2.1. These findings establish that AS-IV protects spermatocytes through coordinated cGAS-STING suppression and Nrf2-dependent HMOX1 activation. Causal necessity of cGAS-STING activation was confirmed by RU.521-mediated pathway blockade, and direct AS-IV binding to cGAS (KD = 0.619 μM) and STING (KD = 0.097 μM) was validated by surface plasmon resonance.

Microplastics and nanoplastics are ubiquitous across global environments and can bioaccumulate through food chains, arousing concerns over their links to rising rates of male infertility. Relevant literature was retrieved from PubMed, Web of Science Core Collection and Scopus, and eligible original human, animal and cell studies were screened and analyzed. Epidemiological data confirm microplastic accumulation in human reproductive tissues, which correlates with declining semen quality. Experimental evidence reveals that microplastics penetrate the blood-testis barrier to trigger testicular oxidative stress, DNA damage and epigenetic dysregulation, further causing inflammation, barrier disruption, hormone imbalance and sustained spermatogenic injury. This review systematically interprets core toxic cascades of microplastic-induced male infertility, and compares heterogeneous reproductive toxicity from varying particle sizes, polymers, doses and combined pollutants. Research gaps and future directions are also outlined, providing theoretical support for assessing male reproductive hazards of microplastic pollution.

Methotrexate (MTX) is a widely used chemotherapeutic and immunosuppressive agent; however, its clinical use is limited by severe testicular toxicity associated with oxidative stress, inflammation, apoptosis, and ferroptosis. Therefore, the present study investigated the protective effects of icariin-loaded nanoliposomal formulation (ICA-LNPs) against MTX-induced testicular injury in rats compared with crude icariin (ICA). Sixty adult male Sprague Dawley rats were randomly allocated into six groups: control, ICA, ICA-LNPs, MTX, MTX + ICA, and MTX + ICA-LNPs. MTX administration markedly impaired reproductive function, evidenced by significant reductions in testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), sperm count, motility, and viability, together with increased sperm abnormalities. MTX also disrupted the NRF2/HO-1/NQO1 signaling pathway, suppressed antioxidant defenses, and elevated oxidative stress markers. In addition, MTX significantly activated NF-κB-mediated inflammatory responses, enhanced apoptotic and ferroptotic pathways, increased iron accumulation and ACSL4 expression, and reduced GPX4 and SLC7A11 expression in testicular tissue. Histopathological examination further confirmed severe degeneration of seminiferous tubules and impaired spermatogenesis. Treatment with ICA-LNPs significantly ameliorated these alterations and demonstrated superior protective efficacy compared with crude ICA. ICA-LNPs restored reproductive hormones, improved semen quality, enhanced antioxidant status, suppressed inflammatory mediators, reduced apoptosis and ferroptosis-related markers, and preserved normal testicular histoarchitecture. Furthermore, GPX4 immunoreactivity was markedly increased, whereas ACSL4 expression was significantly reduced following ICA-LNPs treatment. In conclusion, ICA-loaded nanoliposomal formulation effectively attenuated MTX-induced testicular toxicity and was associated with reduced oxidative stress, inflammation, apoptosis, and ferroptosis, highlighting its potential as a promising therapeutic strategy for preserving male reproductive function.

N-glycolylneuraminic acid (Neu5Gc), a dietary xenobiotic sialic acid acquired primarily from red meat and dairy products, cannot be synthesized by humans due to the inactivation of the CMAH gene. In contrast, its endogenous homologue N-acetylneuraminic acid (Neu5Ac) is abundant in human reproductive tissues. Although anti-Neu5Gc antibodies have been associated with infertility, the direct impact of free sialic acids on sperm function remains unclear. This study investigated the dose-dependent effects of physiologically relevant concentrations (0-10 μM) of Neu5Gc and Neu5Ac on human sperm function in vitro and elucidated the underlying mechanisms. After treatment, key sperm functions were comprehensively evaluated: motility by computer-assisted analysis, penetration capacity through methylcellulose, and acrosome reaction (AR) via chlorotetracycline staining. Sperm ATP concentration and mitochondrial membrane potential were evaluated by ELISA and JC-1. Intracellular Ca[2 +] levels and protein tyrosine phosphorylation were measured fluorometrically and by western blotting, respectively. Both Neu5Gc and Neu5Ac significantly and dose-dependently inhibited sperm total motility, progressive motility, penetration capacity, and both spontaneous and progesterone-induced AR. Neu5Gc and Neu5Ac compromised sperm ATP generation without impairing mitochondrial membrane potential. Mechanistically, these impairments were accompanied by a marked suppression of total protein tyrosine phosphorylation and a reduction in resting intracellular Ca[2+] levels. In conclusion, our in vitro results demonstrate that free Neu5Ac and Neu5Gc, at physiologically relevant micromolar concentrations, impair essential sperm functions by disrupting capacitation-associated tyrosine phosphorylation and calcium signaling pathways, suggesting a possible diet-environment-reproductive health axis in male fertility.

Propylparaben is an antimicrobial preservative commonly found in consumer products. Recent studies suggest that it has endocrine-disrupting properties and may adversely affect female reproductive health. However, it remains unclear whether short-term propylparaben exposure affects the ovaries and oviducts of adult females. This study examined the effects of 10 days of exposure to environmentally relevant levels of propylparaben on selected genes, hormone levels, and protein expression in the murine ovary and oviduct. Adult CD-1 mice (n = 6/per group) were orally administered vehicle control (corn oil) or propylparaben at 20 μg/kg/day, 200 μg/kg/day, or 20 mg/kg/day by dispensing the dosing solution from a pipette tip into the oral cavity. After dosing, sera, ovaries, and oviducts were collected. Serum was used to measure progesterone and estradiol levels. Ovaries and oviducts were analyzed for the expression of steroidogenic regulators, apoptotic factors, antioxidant enzymes, epithelial cell markers, and estrogen receptor alpha. Differences between propylparaben-treated groups and controls were evaluated statistically. In the ovary, propylparaben selectively increased mRNA levels of Cyp17a1 (20 mg/kg/day), Cyp19a1 (20 mg/kg/day), and Bcl2 (20 and 200 μg/kg/day) compared with controls (p < 0.05). In the oviduct, propylparaben selectively increased mRNA levels of Bcl2 (20 and 200 μg/kg/day), Bax (all groups), and Gpx1 (20 mg/kg/day), and decreased protein levels of the ciliated cell marker acetylated-α-tubulin (20 μg/kg/day) compared with controls (p < 0.05). Last, hormone levels were similar across treatment groups (p > 0.05). These findings suggest that short-term propylparaben exposure alters key regulators in the ovary and oviduct, yet the broader implications for female reproductive health remain unclear.

Triptolide (TP), the principal bioactive component of Tripterygium wilfordii (T. wilfordii), exhibits potent pharmacological activities; however, its clinical application is limited by severe male reproductive toxicity, and effective interventions to mitigate reproductive toxicity remain limited. Although the relationship between ferroptosis and TP-induced male reproductive damage has garnered attention, it is still not fully understood whether intervening in ferroptosis can serve as a viable approach to ameliorate TP-induced testicular injury. Thus, this study aimed to characterize TP-induced ferroptosis in mouse testes and Sertoli cells and evaluate whether pharmacological inhibition of ferroptosis by liproxstatin-1 (Lip-1) could serve as a protective strategy against TP-induced testicular injury. In this study, we demonstrated that TP exposure caused significant testicular damage, characterized by pathological histological changes, disruption of the blood-testis barrier (BTB) integrity, a decrease in sperm count and motility, and an increase in sperm deformity. Mechanistically, TP downregulated key redox regulators (NRF2, HO-1, SLC7A11, and GPX4) and iron homeostasis proteins (FTH1, FTL, and FPN), implicating ferroptosis in male reproductive toxicity. Crucially, co-treatment with the Lip-1 effectively mitigated both BTB disruption and testicular damage induced by TP, both in vivo and in vitro. The protective effect was associated with the attenuation of lipid peroxidation and the restoration of the NRF2/HO-1/SLC7A11/GPX4 pathway. Collectively, our findings suggested that pharmacological inhibition of ferroptosis by Lip-1 represented a promising strategy against TP-induced BTB disruption and testicular injury.

Global sperm concentration has declined by over 50% in the past five decades, with environmental pollutants identified as a critical driving factor. This review focuses on testicular-level evidence regarding the effects of microplastics, bisphenol compounds, and air pollution on male reproductive health, with an emphasis on spermatogenic and testicular somatic cell mechanisms. Microplastics have been detected in human testicular tissue, with a causal pathway established linking gut microbiota dysbiosis, immune activation, and blood-testis barrier disruption. Bisphenol A substitutes show comparable or greater reproductive toxicity than BPA, with BCAT1-mediated ferroptosis identified as a novel testicular injury mechanism. For air pollution, spermatogenesis stage I (70-90 days prior to semen collection) has been pinpointed as the critical PM2.5 exposure vulnerability window, with metal constituents of traffic-derived PM2.5 as the primary toxicity drivers. Future research should prioritize prospective cohort studies and clinical intervention trials targeting these emerging mechanistic pathways.

Allura Red AC, a widely used synthetic azo dye in food and beverages, lacks comprehensive data on developmental toxicity across exposure levels-prompting this evaluation of developmental toxicity in Sprague-Dawley (SD) rats per standardized guidelines. Fifty pregnant rats were randomly assigned to oral gavage treatments: Allura Red AC at 40, 200, or 1000 mg/kg/day, distilled water (negative control), or 300 mg/kg aspirin (positive control) from gestation day (GD) 6-15, a critical window for organogenesis. Maternal and fetal parameters were assessed on GD 20.The positive control group showed significant embryotoxicity, validating the test system's sensitivity. Notably, Allura Red AC at ≥ 40 mg/kg caused a statistically significant reduction in fetal posterior fontanel width vs. the negative control-hinting at effects on cranial bone ossification. No adverse changes emerged in maternal reproductive metrics (e.g., weight gain, implantation sites) or key fetal indicators (viability, body size, sex ratio, external/visceral/skeletal malformations). Under this study's conditions, Allura Red AC induced no overall adverse developmental toxicity, and no teratogenic structural malformations but may alter specific cranial ossification patterns at doses ≥ 40 mg/kg. However, this finding cannot prove that Allura Red AC fails to change cranial ossification patterns at doses below 40 mg/kg.

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.