Congenital heart disease is a leading cause of death in newborns, yet many of its molecular mechanisms remain unknown. Both maternal obesity and diabetes increase the risk of congenital heart disease in offspring, with recent studies suggesting these conditions may have distinct teratogenic mechanisms. The global prevalence of obesity is rising, and while maternal obesity is a known risk factor for fetal congenital heart disease, the specific mechanisms are largely unexplored.

We used a murine model of diet-induced maternal obesity, without diabetes, to produce dams that were overweight but had normal blood glucose levels. Embryos were generated and their developing hearts analyzed. Transcriptome analysis was performed using single-nucleus and bulk RNA sequencing. Global and phospho-enriched proteome analysis was performed using tandem mass tag-mass spectroscopy. Immunobloting and histologic evaluation were also performed. Analysis revealed disrupted oxidative phosphorylation and reactive oxygen species formation, with reduced antioxidant capacity, evidenced by downregulation of genes Sod1 and Gp4x, and disrupted Hif1a signaling. Evidence of oxidative stress, cell death signaling, and alteration in Rho GTPase and actin cytoskeleton signaling was also observed. Genes involved in cardiac morphogenesis, including Hand2, were downregulated, and fewer mature cardiomyocytes were present. Histologic analysis confirmed increased cardiac defects in embryos exposed to maternal obesity.

These findings demonstrate that maternal obesity alone can result in cardiac defects through mechanisms similar to those associated with maternal hyperglycemia. This study provides valuable insight into the role of maternal obesity, a growing and modifiable risk factor, in the development of the most common birth defect, congenital heart disease.

Infertility has emerged as a significant global health concern, affecting nearby 8-12% of couples in reproductive age worldwide. Increasing evidence suggests a potential link between human papillomavirus (HPV) and infertility in both men and women. Some research indicate that HPV can infect various components of semen, potentially affecting sperm quality by decreasing motility, viability, and increasing DNA fragmentation, all of which may contribute to male infertility. The virus can attach to the equatorial region of the sperm head, enabling infected sperm to transmit the virus to the oocyte or placenta. Consequently, HPV potentially induces apoptosis in trophoblastic cells and disrupts their adhesion to endometrial cells, which raises the risk of miscarriage. HPV may also affect ovarian reserve by causing chronic inflammation, which can impair granulosa cell function and lower serum anti-Müllerian hormone (AMH) levels. Besides, HPV-related immune responses also contribute to infertility by producing anti-sperm antibodies (ASAs), which cause sperm clumping, reduce motility through cervical mucus, activate the complement system that damages sperm in the female reproductive tract and interfere with sperm-egg interactions. Moreover, HPV infection has been linked to reduced success rates in assisted reproductive technologies (ART), potentially disrupting critical processes such as the acrosome reaction, sperm-oocyte interaction, and fusion. One potential mechanism through which HPV contributes to infertility is oxidative stress (OS). Triggered OS can negatively impact sperm quality and cause damage to the female reproductive system, ultimately contributing to infertility. Despite these associations, the precise mechanisms and the strength of the relationship remain uncertain. Thus, this review seeks to investigate the potential impact of HPV on infertility, particularly its effects on the reproductive system through OS. A clearer understanding of these processes could inform future health strategies for addressing HPV-related infertility.

Valproic acid (VPA) is a very effective therapy used to treat generalized epilepsy, but it must be avoided during pregnancy as it leads to a high risk of teratogenesis. Its teratogenic effect is believed to be due to its placental toxic effect, altering angiogenesis and inducing oxidative stress. Piracetam (PIRA) is a derivative of the neurotransmitter γ-aminobutyric acid (GABA) and has anti-oxidative and pro-angiogenic features. However, its effects against Valproic acid-evoked placental toxicity and abnormal fetal development have not been mechanistically examined. Herein, the present study targets angiogenesis and oxidative stress by Piracetam to investigate the potential modulation of Valproic acid-induced placental toxicity and abnormal fetal development in rats. After administration of Valproic acid (500 mg/kg/day, orally) and/or piracetam (500 mg/kg/day, orally) from the 6th to 15th of gestation, fetuses and placenta were obtained for analysis. The present findings revealed that Piracetam improved the histopathological lesions in the placenta and restored the labyrinth zone area percent. Moreover, it improved the intra-uterine growth retardation (IUGR) via restoring fetal body weight and length and also ameliorated all external malformations (subcutaneous hemorrhage, fore limb, and hind limb anomalies) and additionally amended the skeletal lack of ossification. These favorable effects of Piracetam were mediated by the enhancement of placental angiogenesis via the VEGF/eNOS/TGF-β pathway and attenuating placental oxidative stress, which appeared as decreased MDA content and increased GSH and TAC levels. In conclusion, activation of placental angiogenesis via the VEGF/eNOS/TGF-β axis alongside inhibition of oxidative stress by Piracetam can ameliorate Valproic acid-evoked placental toxicity and, subsequently, fetal malformations in rats.

Embryonic development is a complex process of concurrent events comprising cell proliferation, differentiation, morphogenesis, migration, and tissue remodeling. To cope with the demands arising from these developmental processes, cells increase their nutrient uptake, which subsequently increases their metabolic activity. Mitochondria play a key role in the maintenance of metabolism and production of reactive oxygen species (ROS) as a natural byproduct. Regulation of ROS by antioxidants is critical and tightly regulated during embryonic development, as dysregulation results in oxidative stress that damages essential cellular components such as DNA, proteins, and lipids, which are crucial for cellular maintenance and in extension development. However, during development, exposure to certain exogenous factors or damage to cellular components can result in an imbalance between ROS production and its neutralization by antioxidants, leading to detrimental effects on the developmental process. In this review article, we highlight the crucial role of redox homeostasis in normal development and how disruptions in redox balance may result in developmental defects.

Sows and piglets face heightened oxidative stress during gestation and lactation, yet strategies to simultaneously mitigate these challenges remain underexplored. This study investigated the effects of β-carotene and superoxide dismutase (SOD) supplementation on 140 Landrace × Yorkshire sows (parity 3-5) randomly assigned to (1) a control; (2) long-term low-dose treatment (25 mg/kg β-carotene, 4 mg/kg SOD, or both) throughout gestation-lactation; or (3) short-term high-dose treatment (100 mg/kg β-carotene, 14 mg/kg SOD, or both) administered 7 days pre/post-weaning and farrowing. Our data indicate that the antioxidants enhanced the productive performance of both sows and piglets, with the most pronounced effect observed in the long-term, low-dose combined administration of β-carotene and SOD. The composite antioxidants significantly improved the systemic antioxidant capacity in sows, while concurrently reducing the cortisol and lipopolysaccharide concentrations in the serum. This enhancement contributed to elevations in serum progesterone and prolactin levels at day 40 of gestation and farrowing, respectively, ultimately increasing the number of weaned piglets and decreasing the backfat loss. In addition, the compound antioxidants improved the serum antioxidant indices of piglets, increased the growth hormone concentrations, and improved the litter weight gain. Mechanistically, the placental upregulation of CAT, GPX1, and GLUT3, alongside Claudin1, Occludin, and ZO-1 expression, underpinned improved nutrient transport and barrier function. These findings demonstrate that β-carotene and SOD synergistically transfer antioxidant capacity via placental and colostrum pathways, offering a viable strategy for integrated sow-piglet management.

This study was aimed to investigate the effects of ozone (O3) treatment during incubation period (IP) on hatchability, hatch window, chick quality and organ growth, bacterial load of feces and first-week growth performance in broilers. A total of 240 hatching eggs were weighed and randomly divided into control group (O3-IP (-)) and O3 treatment (O3-IP (+)). A commercial O3 generator was placed into the setter and O3 treatment (at the level of 0.050 ppm) was applied during 1 min per hour in a cyclic period of 3 days during the 18-day incubation period. The egg weight loss between 1 and 18 days ranged with values 8.59% in O3-IP (-) and 10.63% in O3-IP (+) group. The pipping time and incubation length was determined as 500.67 h and 527.33 h in O3-IP (-) and 489.67 h and 518.33 h in O3-IP (+) respectively. The yolk sac weight was found to be higher in the O3-IP (-) group compared to the O3-IP (+). In conclusion, O3 treatment during incubation period seems to be cause an acceleration for pipping time and shortening of total incubation period, unsteady effects for chick growth and quality, inhibitory effect for bacterial growth in feces.

Endometriosis significantly impacts the physical and mental health of women of reproductive age. While some patients can achieve pregnancy through surgery or in vitro fertilization (IVF), many still struggle with IVF failure due to poor oocyte quality. This presents a major clinical challenge that requires immediate attention. The causes of oocyte quality decline in endometriosis patients are diverse and have not yet been definitively identified. Furthermore, effective diagnostic mechanisms and therapeutic strategies remain elusive. To provide possible clinical solutions to improve pregnancy rates in patients with endometriosis, this review evaluates the current literature on the impact of endometriosis on oocyte quality, the possible mechanisms and management strategies of endometriosis leading to poor oocyte quality.

Vitrification is widely used in assisted reproductive technology (ART) for female infertility, but the long-term effect on the embryo of vitrification has not been comprehensively studied. The study aimed to investigate the effect of vitrification on long-term development of mouse embryos. The warmed embryos which were frozen at 8-cell stage were cultured in vitro until the blastocyst stage and were transferred into recipients. Immunofluorescence staining was performed to evaluate the reactive oxygen species (ROS) level, mitochondrial function, cell apoptosis, DNA damage and histone epigenetic modification in blastocysts. Transmission electron microscopy (TEM) analysis was performed to exam the mitochondrial ultrastructure in blastocysts. The related gene expression and transcriptome profiles were investigated by RT-qPCR and RNA-seq, respectively. Blastocyst and implantation frequencies were not significantly affected by vitrification. However, vitrification significantly reduced blastocyst cell number and the live pup frequency. Vitrification induced ROS accumulation, DNA damage, and apoptosis in mouse blastocysts. The homologous recombination (NHEJ) is the major DNA repair pathway for vitrified embryos. Vitrification elevated H3K4me2/3, H4K12ac, and H4K16ac levels and reduced m6A modification in blastocysts. Moreover, vitrification significantly altered transcriptome profiles of mice placentas and brains at embryonic day 18.5 (E18.5). Thus, vitrification exhibited a long-term effect on mouse embryo viability by increasing ROS levels, DNA damage, altering the epigenetic modifications and transcriptome profiles.

Oxidative stress (OS) induced by an imbalance in reactive oxygen species (ROS) levels in vitro impairs embryonic development. Here, we assessed the effects of alpha-lipoic acid (ALA) in in vitro production media on OS reduction, embryonic development, and cryotolerance of bovine embryos. We evaluated the effects of adding different concentrations of ALA (2.5, 5, 10, and 25 μM) to in vitro maturation (IVM) or in vitro culture (IVC) medium on embryonic development. We also determined the effects of adding ALA (25 μM) to the IVM and IVC medium in the same routine on the development and quality of embryos, ROS levels, and cryotolerance. Embryos were produced in vitro using conventional protocols for each treatment. The inclusion of ALA in the IVM and IVC media did not affect the development or quality of embryos; however, it reduced ROS levels in grade II embryos and increased hatching after 12 h on day 7 in grade I embryos and on day 8 in grade II embryos after warming. These findings prompt questions regarding the potential of ALA in improving embryo metabolism, considering the initial embryo recovery in the first few hours of embryo warming.

As one of Mexico's most crucial water storage facilities, the Villa Victoria Reservoir (VVR) supplies water to over six million people residing in the Mexico City Metropolitan Area. In recent years, this water resource has been subjected to significant risks due to several factors, including human population growth, alterations in global climate patterns, excessive resource utilization, and insufficient protective regulations, thereby endangering not only the biocenosis itself, but also the water supply for numerous inhabitants. This study aimed to evaluate the current state of the reservoir through the determination of conventional and emerging pollutants present in the sampling points, as well as embryotoxicity and oxidative damage in Danio rerio embryos exposed to effluents from the VVR. Embryotoxicity was quantified using the General Morphology Score (GMS) and teratogenic index, whereas oxidative damage was assessed based on lipid peroxidation, hydroperoxide content, oxidized proteins, antioxidant enzyme activity, and gene expression. These results revealed the presence of heavy metals, diverse pharmaceutical compounds, and pesticides. In addition, elevated lipid, hydroperoxide, and protein oxidation accompanied by alterations in superoxide dismutase (SOD) and catalase (CAT) enzymatic activity were observed during exposure. GMS resulted in impaired embryo development and teratogenic effects, including pericardial, axial, and skeletal edema. Furthermore, the upregulation of genes associated with apoptotic processes and antioxidant defense reflects a comprehensive response to oxidative stress. The study concluded that pollutants in VVR water induced oxidative damage, modified antioxidant activity, elicited embryotoxicity, and upregulated oxidative damage-related genes. The findings underscore the necessity of undertaking restoration efforts for water sources, as pollution can potentially endanger aquatic organisms and human well-being.

The association between embryo morphokinetics and its developmental competence is well documented. For instance, early cleaved embryos are more competent in developing to blastocysts, whereas the proportion of abnormally cleaved embryos that further developed to blastocysts is low. Numerous factors, such as the parental age, lifestyle, health, and smoking habits have been reported to affect the embryo morphokinetics and, consequently, its development. However, less is known about the effect of environmental stressors on embryo morphokinetics. The current review discusses the effect of the most concerning environmental stressors on embryo morphokinetics. These stresses include heat stress and human-made chemicals such as phthalates (e.g., bis-(2-ethylhexyl phthalate, dibutyl phthalate, dimethyl phthalate, and their primary metabolites), herbicides (e.g., diaminochlorotriazine, the primary metabolite of atrazine), pharmaceutical compounds (e.g., carbamazepine, nocodazole) and pro-oxidant agents (cumene hydroperoxide, Triton X-100), as well as naturally occurring toxins such as mycotoxin (e.g., aflatoxin B1 and its metabolite, and ochratoxin A). In addition, this review discusses the effect of ionizing or non-ionizing radiation and viral infections (e.g., SARS-CoV-2, papillomavirus). Finally, it points out some potential mechanisms that underlie the impairment of embryo morphokinetics, and it suggests protective compounds, mainly the supplementation of antioxidants to improve the morphokinetics, and consequently, the embryo developmental competence.

Our research aims to investigate the specific mechanisms by which copper inhibits the asexual proliferation of Aurelia coerulea polyps.

Aurelia coerulea polyps were exposed to various CuSO4 concentrations to study metamorphosis and budding proliferation. Oxidative stress markers (ROS, MDA, CAT, H2O2, T-AOC, SOD) were measured in polyps and early strobilae. Transcriptomic analysis were used to compare differences in gene expression and enrichment pathways between untreated and copper-exposed polyps. Additionally, RT-qPCR was used to analyze the expression of key molecules. Antioxidant L-Ascorbic acid was applied to determine the role of oxidative stress in asexual reproduction of Aurelia coerulea polyps when exposed to copper.

Copper inhibited strobilization and budding of Aurelia coerulea polyps in a dose-dependent manner, in which oxidative stress was involved. Transcriptomic data suggested that the DNA replication pathway was significantly enriched in early strobilae compared to polyps. However, copper treatment repealed the difference of DNA replication pathway between early strobilae compared and polyps. Transcriptomic data suggested that alanine, aspartate, and glutamate metabolism pathways were enriched in untreated budding polyps compared to copper-exposed polyps. After applying the antioxidant L-Ascorbic acid to copper-exposed polyps, various oxidative indicators changed to different extents, with increases in ROS, MDA, CAT, H2O2, and SOD and a decrease in T-AOC. Further more, the time required for polyps to develop into early strobila was shortened, indicating that the delay in metamorphosis caused by copper exposure was effectively alleviated. And the budding rate increased, indicating that the inhibition of budding proliferation caused by copper exposure was effectively alleviated. The expression of key genes were consist with the transcriptomic sequencing results.

Copper exposure causes oxidative stress resulting in the inhibition of asexual reproduction in Aurelia coerulea polyps, including metamorphosis and budding.

Melatonin is a hormone mainly secreted by the pineal gland during the circadian cycle, with low levels during the daytime and prominent levels during the night. It is involved in numerous physiological functions including the immune system, circadian rhythm, reproduction, fertilization, and embryo development. In addition, melatonin exerts anti-inflammatory and antioxidant effects inside the body by scavenging reactive oxygen and reactive nitrogen species, increasing antioxidant defenses, and blocking the transcription factors of pro-inflammatory cytokines. Its protective activity has been reported to be effective in various reproductive biotechnological processes, including in vitro maturation (IVM), embryo development, and survival rates. In this comprehensive review, our objective is to summarize and debate the potential mechanism and impact of melatonin on oocyte maturation and embryo development through various developmental routes in different mammalian species.

Studies have shown cerebrovascular dysfunction in offspring with full-gestational electronic cigarette (Ecig) exposure, but little is known about how individual trimester exposure impacts offspring health. This study aimed to determine if there is a critical window during gestation that contributes to vascular and anxiety-like behavioural changes seen with full-term exposure. To test this, rats were time-mated, and the pregnant dams were randomly assigned to Ecig exposure during first trimester (gestational day, GD2-7), second trimester (GD8-14), third trimester (GD15-21) or full-term gestation (GD2-21). We also assessed the effect of maternal preconception exposure. Both male and female offspring from all maternal exposure conditions were compared to offspring from dams under ambient air (control) conditions. Ecig exposure consisted of 60-puffs/day (5 days/week) using either 5 or 30 watts for each respective exposure group. We found that maternal exposure to Ecig in the second and third trimesters resulted in a decrease (23-38%) in vascular reactivity of the middle cerebral artery (MCA) reactivity in 3- and 6-month-old offspring compared to Air offspring. Further, the severity of impairment was comparable to the full-term exposure (31-46%). Offspring also displayed changes in body composition, body mass, anxiety-like behaviour and locomotor activity, indicating that Ecigs influence neurodevelopment and metabolism. Maternal preconception exposure showed no impact on offspring body mass, anxiety-like behaviour, or vascular function. Thus, the critical exposure window where Ecig affects vascular development in offspring occurs during mid- to late-gestation in pregnancy, and both 5 W and 30 W exposure produce significant vascular dysfunction compared to Air. KEY POINTS: Exposure to electronic cigarettes (Ecigs) is known to increase risk factors for cardiovascular disease in both animals and humans. Maternal Ecig use during pregnancy in rodents is found to impair the vascular health of adolescent and adult offspring, but the critical gestation window for Ecig-induced vascular impairment is not known. This study demonstrates Ecig exposure during mid- and late-gestation (i.e. second or third trimester) results in impaired endothelial cell-mediated dilatation (i.e. middle cerebral artery reactivity) and alters anxiety-like behaviour in offspring. Maternal exposure prior to conception did not impact offspring's vascular or anxiety-like behavioural outcomes. Rodent models have been a reliable and useful predictor of inhalation-induced harm to humans. These data indicate maternal use of Ecigs during pregnancy should not be considered safe, and begin to inform clinicians and women about potential long-term harm to their offspring.

5-hydroxymethylfurfural is a common byproduct in food. However, its effect on growth and development remains incompletely understood. This study investigated the developmental toxicity of 5-HMF to Drosophila larvae. The growth and development of Drosophila melanogaster fed with 5-50 mM 5-HMF was monitored, and its possible mechanism was explored. It was found that 5-HMF prolonged the developmental cycle of Drosophila melanogaster (25 mM and 50 mM). After 5-HMF intake, the level of reactive oxygen species in the third instar larvae increased by 1.23-1.40 fold, which increased the level of malondialdehyde and caused changes in antioxidant enzymes. Moreover, the nuclear factor erythroid-2 related factor 2 antioxidant signaling pathway and the expression of heat shock protein genes were affected. At the same time, 5-HMF disrupted the glucose and lipid metabolism in the third instar larvae, influencing the expression level of key genes in the insulin signal pathway. Furthermore, 5-HMF led to intestinal oxidative stress, and up-regulated the expression of the pro-apoptotic gene, consequently impacting intestinal health. In short, 5-HMF causes oxidative stress, disturbs glucose and lipid metabolism and induces intestinal damage, damaging related signaling pathways, and ultimately affecting the development of Drosophila melanogaster.

Biliary atresia (BA) is a cholangiopathy affecting the extrahepatic bile duct (EHBD) of newborns. The etiology and pathophysiology of BA are not fully understood; however, multiple causes of damage and obstruction of the neonatal EHBD have been identified. Initial damage to the EHBD likely occurs before birth. We discuss how different developmental stages in utero and birth itself could influence the susceptibility of the fetal EHBD to damage and a damaging wound-healing response. We propose that a damage-repair response of the fetal and neonatal EHBD involving redox stress and a program of fetal wound healing could-regardless of the cause of the initial damage-lead to either obstruction and BA or repair of the duct and recovery. This overarching concept should guide future research targeted toward identification of factors that contribute to recovery as opposed to progression of injury and fibrosis. Viewing BA through the lens of an in utero damage-repair response could open up new avenues for research and suggests exciting new therapeutic targets.

The relationship between maternal oxidative balance score (OBS) in pregnancy, representing overall oxidative balance status by integrating dietary and lifestyle factors, and congenital heart defects (CHD) remains unclear; therefore, this study attempted to explore their associations among the Chinese population. We conducted a case-control study including 474 cases and 948 controls in Northwest China. Pregnant women were interviewed to report diets and lifestyles in pregnancy by structured questionnaires. Logistic regression models were used to estimate the adjusted ORs (95%CIs). Maternal OBS ranged from 6 to 34 among cases, and 5 to 37 among controls. Comparing the highest with the lowest tertile group, the adjusted OR for CHD was 0.31 (0.19-0.50). The CHD risk was reduced by 7% (OR = 0.93, 95%CI = 0.90-0.95) in association with per 1 higher score of OBS during pregnancy. The inverse relationship between maternal OBS and CHD risk appeared to be more pronounced among participants in urban areas (OR = 0.89, 95%CI = 0.86-0.93). Maternal OBS during pregnancy showed good predictive values for fetal CHD, with the areas under the receiver operating characteristic curve 0.78 (0.76-0.81). These findings highlighted the importance of reducing oxidative stress through antioxidant-rich diets and healthy lifestyles among pregnant women to prevent fetal CHD.

It is widely accepted that humans are constantly exposed to micro-plastics and nano-plastics through various routes, including inhalation of airborne particles, exposure to dust, and consumption of food and water. It is estimated that humans may consume thousand to millions of micro-plastic particles, equating to several milligrams per day. Prolonged exposure to micro-plastics and nano-plastics has been linked to negative effects on different living organisms, including neurotoxicity, gastrointestinal toxicity, nephrotoxicity, and hepatotoxicity, and developmental toxicities. The main purpose of this review is to explore the effect of micro-plastics and nano-plastics on the male and female reproductive system, as well as their offspring, and the associated mechanism implicated in the reproductive and developmental toxicities. Micro-plastics and nano-plastics have been shown to exert negative effects on the reproductive system of both male and female mammals and aquatic animals, including developmental impacts on gonads, gametes, embryo, and their subsequent generation. In addition, micro-plastics and nano-plastics impact the hypothalamic-pituitary axes, leading to oxidative stress, reproductive toxicity, neurotoxicity, cytotoxicity, developmental abnormalities, poor sperm quality, diminishes ovarian ovulation and immune toxicity. This study discusses the so many different signaling pathways associated in the male and female reproductive and developmental toxicity induced by micro-plastics and nano-plastics.

Neonicotinoids (NEOs) are widely used insecticides that are ubiquitous in agricultural use. Since NEOs are found in natural waters as well as in tap water and human urine in regions where NEOs are widely used, NEOs pose a potential hazard to non-target organisms such as animals and humans. Some of the commonly detected NEOs are imidacloprid (IMD), thiamethoxam (TMX), and its metabolite clothianidin (CLO). Although previously published scientific information, including an assessment of the environmental risks, particularly for bees, had resulted in a ban on the outdoor use of these three NEOs in the EU - their use is now only permitted in closed greenhouses - these NEOs continue to be used in agriculture in many other parts of the world. Therefore, a detailed study and comparison of the effects of NEOs on the embryonic development of non-target organisms is needed to further define the risk profiles. Embryos of the South African clawed frog Xenopus laevis, a well-established aquatic model, were exposed to different concentrations of IMD, TMX, or CLO (0.1-100 mg/L) to study and compare the possible effects of a single contaminant in natural water bodies on early embryogenesis. The results included a reduced body length, a smaller orbital space, impaired cranial cartilage and nerves, and an altered heart structure and function. At the molecular level, NEO exposure partially resulted in an altered expression of tissue-specific factors, which are involved in eye, cranial placode, and heart development. Our results suggest that the NEOs studied negatively affect the embryonic development of the non-target organism X. laevis. Since pesticides, especially NEOs, pollute the environment worldwide, it is suggested that they are strictly controlled and monitored in the areas where they are used. In addition, the question arises as to whether pesticide metabolites also pose a risk to the environment and need to be investigated further so that they can be taken into account when registering ingredients.

Although widely known as a tumor suppressor, the breast cancer 1 susceptibility protein (BRCA1) is also important in development, where it regulates fetal DNA repair pathways that protect against DNA damage caused by physiological and drug-enhanced levels of reactive oxygen species (ROS). We previously showed that conditional heterozygous (+/-) knockout (cKO) mouse embryos with a minor 28% BRCA1 deficiency developed normally in culture, but when exposed to the ROS-initiating drug, alcohol (ethanol, EtOH), exhibited embryopathies not evident in wild-type (+/+) littermates. Herein, we characterized a directBrca1 +/- knockout (KO) model with a 2-fold greater (58%) reduction in BRCA1 protein vs. the cKO model. We also characterized and compared learning & memory deficits in both the cKO and KO models. Even saline-exposed Brca1 +/- vs. +/+ KO progeny exhibited enhanced oxidative DNA damage and embryopathies in embryo culture and learning & memory deficits in females in vivo, which were not observed in the cKO model, revealing the potential pathogenicity of physiological ROS levels. The embryopathic EtOH concentration for cultured direct KO embryos was half that for cKO embryos, and EtOH affected Brca1 +/+ embryos only in the direct KO model. The spectrum and severity of EtOH embryopathies in culture were greater in both Brca1 +/- vs. +/+ embryos, and direct KO vs. cKO +/- embryos. Motor coordination deficits were evident in both male and female Brca1 +/- KO progeny exposed in utero to EtOH. The results in our direct KO model with a greater BRCA1 deficiency vs. cKO mice provide the first evidence for BRCA1 protein dose-dependent susceptibility to developmental disorders caused by physiological and drug-enhanced oxidative stress.

Studying placental functions is crucial for understanding pregnancy complications. However, imaging placenta is challenging due to its depth, volume, and motion distortions. In this study, we have developed an implantable placenta window in mice that enables high-resolution photoacoustic and fluorescence imaging of placental development throughout the pregnancy. The placenta window exhibits excellent transparency for light and sound. By combining the placenta window with ultrafast functional photoacoustic microscopy, we were able to investigate the placental development during the entire mouse pregnancy, providing unprecedented spatiotemporal details. Consequently, we examined the acute responses of the placenta to alcohol consumption and cardiac arrest, as well as chronic abnormalities in an inflammation model. We have also observed viral gene delivery at the single-cell level and chemical diffusion through the placenta by using fluorescence imaging. Our results demonstrate that intravital imaging through the placenta window can be a powerful tool for studying placenta functions and understanding the placental origins of adverse pregnancy outcomes.

To investigate curcumin (CUR) as the protector against the harmful effects of low-frequency electromagnetic field(LF- EMF, 50 Hz) during pregnancy period, 5 males and 15 females of Wistar rat mated and vaginal plaques were observed. Then, the pregnant rats were divided into six groups. During pregnancy(21 days), the EMF group was exposed to EMF for 30 min/day, the CUR group received a single dose of 50 mg/kg/daily CUR intraperitoneal, the EMF+CUR group was injected CUR and exposed to EMF daily. The DMSO(dimethyl sulfoxide) group was injected solvent of CUR (DMSO) intraperitoneal with the same volume of CUR solvent, the sham group was placed through the solenoid in the same conditions as the first group without exposure and the control group was kept in their cage in normal condition. After four weeks, babies born were divided according to the mother groups and sacrificed. Then, the three tissues injuries were investigated. EMF exposure led to an increase in outstanding necrotic areas in hippocampal tissue, an increase in the amount of hyperemia(p = 0.017) and necrotic(p = 0.005) in kidneys, and degeneration in liver tissue(p = 0.007) in the EMF group compared with EMF+CUR groups. A single dose of CUR daily during pregnancy can protect these tissues from injuries caused by LF-EMF exposure in rat fetuses.

Malrotation of the intestine is a prevalent birth anomaly, the etiology of which remains poorly understood. Here, we show that late-stage exposure of Xenopus embryos to atrazine, a widely used herbicide that targets electron transport chain (ETC) reactions, elicits intestinal malrotation at high frequency. Interestingly, atrazine specifically inhibits the cellular morphogenetic events required for gut tube elongation, including cell rearrangement, differentiation and proliferation; insufficient gut lengthening consequently reorients the direction of intestine rotation. Transcriptome analyses of atrazine-exposed intestines reveal misexpression of genes associated with glycolysis and oxidative stress, and metabolomics shows that atrazine depletes key glycolytic and tricarboxylic acid cycle metabolites. Moreover, cellular bioenergetics assays indicate that atrazine blocks a crucial developmental transition from glycolytic ATP production toward oxidative phosphorylation. Atrazine-induced defects are phenocopied by rotenone, a known ETC Complex I inhibitor, accompanied by elevated reactive oxygen species, and rescued by antioxidant supplementation, suggesting that malrotation may be at least partly attributable to redox imbalance. These studies reveal roles for metabolism in gut morphogenesis and implicate defective gut tube elongation and/or metabolic perturbations in the etiology of intestinal malrotation.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions.

Embryo vitrification is a standard procedure in assisted reproductive technology. Previous studies have shown that frozen embryo transfer is associated with an elevated risk of adverse maternal and neonatal outcomes. This study aimed to explore the effects of mouse blastocyst vitrification on the phenotype of vitrified-warmed blastocysts, their intrauterine and postnatal development, and the long-term metabolic health of the derived offspring. The vitrified-warmed blastocysts (IVF + VT group) exhibited reduced mitochondrial activity, increased apoptotic levels, and decreased cell numbers when compared to the fresh blastocysts (IVF group). Implantation rates, live pup rates, and crown-rump length at E18.5 were not different between the two groups. However, there was a significant decrease in fetal weight and fetal/placental weight ratio in the IVF + VT group. Furthermore, the offspring of the IVF + VT group at an age of 36 weeks had reduced whole energy consumption, impaired glucose and lipid metabolism when compared with the IVF group. Notably, RNA-seq results unveiled disturbed hepatic gene expression in the offspring from vitrified-warmed blastocysts. This study revealed the short-term negative impacts of vitrification on embryo and fetal development and the long-term influence on glucose and lipid metabolism that persist from the prenatal stage into adulthood in mice.

Recent years have seen an increased interest in the role of oxidative stress (OS) in pregnancy. Pregnancy inherently heightens susceptibility to OS, a condition fueled by a systemic inflammatory response that culminates in an elevated presence of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the circulatory system. The amplified OS in pregnancy can trigger a series of detrimental outcomes such as underdevelopment, abnormal placental function, and a host of pregnancy complications, including pre-eclampsia, embryonic resorption, recurrent pregnancy loss, fetal developmental anomalies, intrauterine growth restriction, and, in extreme instances, fetal death. The body's response to mitigate the uncontrolled increase in RNS/ROS levels requires trace elements that take part in non-enzymatic and enzymatic defense processes, namely, copper (Cu), zinc (Zn), manganese (Mn), and selenium (Se). Determination of ROS concentrations poses a challenge due to their short half-lives, prompting the use of marker proteins, including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and glutathione (GSH). These markers, indicative of oxidative stress intensity, can offer indirect assessments of pregnancy complications. Given the limitations of conducting experimental studies on pregnant women, animal models serve as valuable substitutes for in-depth research. This review of such models delves into the mechanism of OS in pregnancy and underscores the pivotal role of OS markers in their evaluation.

Phthalate esters (PAEs) are widely used as plasticizers. PAEs are ubiquitous in natural water bodies, with dibutyl phthalate (DBP) being one of the most common PAEs. DBP is prone to leaching or migration into the environment, posing serious health and environmental risks. Carbon nanotubes (CNTs) have been widely used in various fields with the rapid development of nanotechnology. CNTs could alter the environmental behavior and toxicity of co-existing pollutants. CNTs have been shown to rapidly adsorb PEAs. However, current knowledge about the effects of CNTs on DBP toxicity is limited. Here we show that the toxic effects of single and combined exposure to DBP (0.1, 0.5, 1.0 mg/L) and different CNTs (MWCNTs/MWCNTs-COOH, 0.5 mg/L) on the early growth stage of zebrafish. The results suggested that a significant increase in heart rate and heart malformation rate was observed after co-exposure of DBP and MWCNTs/MWCNTs-COOH (p < 0.05). Furthermore, combined exposure increased antioxidant enzyme activity during early developmental stages in zebrafish (p < 0.05). The qRT-PCR results revealed that DBP and MWCNTs/MWCNTs-COOH co-exposure significantly interfered with the expression of genes related to oxidative stress, energy metabolism, development of cardiac function, and apoptosis (p < 0.05). In addition, for oxidative stress and cardiotoxicity, MWCNTs/MWCNTs-COOH aggravated the toxic effects of 0.5 mg/L DBP on embryos/larvae. The metabolomics results showed that co-exposure mitigated the disturbance of amino acid metabolism mediated by single DBP exposure. In general, MWCNTs/MWCNTs-COOH increased the impact of DBP in the early developmental stages of zebrafish. This study provides new insights into the toxicology of early developmental stages of aquatic organisms exposed to co-exist pollutants of DBP and CNTs.

In recent years and as a result of the Covid-19 pandemic, the consumption of dexamethasone (DXE) has increased. This favors that this corticosteroid is highly released in aquatic environments, generating deleterious effects in aquatic organisms. The information on the toxic effects of DXE in the environment is still limited. Thus, the objective of this work was to determine whether DXE at short-term exposure can cause alterations to embryonic development and alteration of oxidative stress-related gene expression patterns in Cyprinus carpio. For this purpose, common carp embryos (2 hpf) were exposed to realistic concentrations of DXE until 96 hpf. Alterations to embryonic development were evaluated at 12, 24, 48, 72 and 96 hpf. In addition, oxidative stress in carp embryos at 72 and 96 hpf was evaluated by cellular oxidation biomarkers (lipoperoxidation level, hydroperoxide and carbonyl protein content) and antioxidant enzymes activities (superoxide dismutase and catalase). Oxidative stress-related gene expression (sod, cat and gpx1) was also evaluated. Our results showed that DXE concentrations above 35 ng/L are capable of producing alterations to embryonic development in 50 % of the embryo population. Furthermore, DXE was able to induce alterations such as scoliosis, hypopigmentation, craniofacial malformations, pericardial edema and growth retardation, leading to the death of half of the population at 50 ng/L of DXE. Concerning oxidative stress, the results demonstrated that DXE induce oxidative damage on the embryos of C. carpio. In conclusion, DXE is capable of altering embryonic development and generating oxidative stress in common carp C. carpio.

The breast cancer 1 (Brca1) susceptibility gene regulates the repair of reactive oxygen species (ROS)-mediated DNA damage, which is implicated in neurodevelopmental disorders. Alcohol (ethanol, EtOH) exposure during pregnancy causes fetal alcohol spectrum disorders (FASD), including abnormal brain function, associated with enhanced ROS-initiated DNA damage. Herein, oxidative DNA damage in fetal brains and neurodevelopmental disorders were enhanced in saline-exposed +/- vs. +/+ Brca1 littermates. A single EtOH exposure during gestation further enhanced oxidative DNA damage, altered the expression of developmental/DNA damage response genes in fetal brains, and resulted in neurodevelopmental disorders, all of which were BRCA1-dependent. Pretreatment with the ROS inhibitor phenylbutylnitrone (PBN) blocked DNA damage and some neurodevelopmental disorders in both saline- and EtOH-exposed progeny, corroborating a ROS-dependent mechanism. Fetal BRCA1 protects against altered gene expression and neurodevelopmental disorders caused by both physiological and EtOH-enhanced levels of ROS formation. BRCA1 deficiencies may enhance the risk for FASD.

The aim of this study was to determine the effect of in ovo co-supplementation of chicken embryos with a multi-strain probiotic containing effective microorganisms and zinc glycine chelate on total antioxidant capacity; concentrations of sulfhydryl groups, bityrosine bridges, formylkynurenines, hydroperoxides, proteins, corticosterone, pro- and anti-inflammatory cytokines and heat shock proteins; and the activity of catalase and superoxide dismutase in the serum, yolk sac and tissues of broiler chickens at 12 h and at 7 days after hatching. The results indicate high SOD activity in the small and large intestines of chicks at 12 h post-hatch in the groups receiving the multi-strain probiotic and in the small intestine and yolk sac of birds receiving the multi-strain probiotic and Zn-Gly chelate. High concentrations of TNF-α and IFN-γ in the yolk sac and serum after in ovo administration of Zn-Gly chelate were observed 12 h after hatching. The use of a probiotic and a probiotic with Zn-Gly chelate increased the total antioxidant capacity in the tissues of chickens. It can be concluded that in ovo administration of a multi-strain probiotic and Zn-Gly chelate can maintain the oxidant/antioxidant balance in chickens and increase the defense capacity against oxidative stress.

Mancozeb (MZ) is widely used as a fungicide in Brazil due to its effectiveness in combating fungal infections in plantations. However, its toxicity to non-target organisms, including aquatic organisms, has been reported in the literature. Recently, Brazilian legislation was updated to allow a concentration of 8 μg/L of MZ in drinking water (Ordinance GM/MS nº 888, of May 4, 2021). However, the safety of this concentration for aquatic organisms has not yet been put to the test. To address this gap, we conducted a study using zebrafish (Danio rerio) embryos at 4 hpf exposed to MZ at the concentration allowed by law, as well as slightly higher sublethal concentrations (24, 72, and 180 μg/L), alongside a control group. We evaluated various morphophysiological markers of toxicity, including survival, spontaneous movements, heart rate, hatching rate, body axis distortion, total body length, total yolk sac area, and total eye area. Additionally, we measured biochemical biomarkers such as reactive oxygen species (ROS) levels, lipid peroxidation, non-protein thiols (NPSH), and mitochondrial bioenergetic parameters. Our results showed that the concentration of 8 μg/L, currently permitted in drinking water according to Brazilian legislation, increased ROS production levels and caused alterations in mitochondrial physiology. Among the markers assessed, mitochondrial bioenergetic function appeared to be the most sensitive indicator of MZ embryotoxicity, as a decrease in complex I activity was observed at concentrations of 8 and 180 μg/L. Furthermore, concentrations higher than 8 μg/L impaired morphophysiological markers. Based on these findings, we can infer that the concentration of MZ allowed in drinking water by Brazilian environmental legislation is not safe for aquatic organisms. Our study provides evidence that this fungicide is a potent embryotoxic agent, highlighting the potential risks associated with its exposure.

Arachidonic acid (AA), an ω-6 polyunsaturated fatty acid involved in signalling pathways that drive cell fate decisions, has an enhancing role in the immunomodulatory effect on mesenchymal stem cells and the vasculogenesis of embryonic stem cells. 3D embryoid bodies (EBs) from pluripotent stem cells (PSCs) have been used as in vitro models for embryotoxicity for various compounds/drugs. Valproic acid (VA), a common anti-epileptic drug, is known to be embryotoxic and cause malformations in embryos. As early embryogenesis depends on AA, we investigated the embryo protective effects of AA against the embryotoxic drug VA in this study. The effects of AA on the proliferation and cell cycle parameters of PSCs were studied. In particular, the potential of AA to abrogate VA-induced embryotoxicity in vitro was evaluated using ROS detection and antioxidant assays. In response to AA, we observed modulation in cell proliferation of induced pluripotent stem cells (iPSCs) and pluripotent NTERA-2 embryonal carcinoma (EC) cells. The present study substantiates the cytoprotective effects of AA against VA. These results imply that AA plays a critical role in the proliferation and differentiation of iPSCs and EC cells and protects the EBs from cytotoxic damage, thereby ensuring normal embryogenesis. Thus, the bioactive lipid AA may be explored for supplementation to benefit pregnant women treated with long-term anti-epileptic drugs to prevent in-utero fetal growth malformations.

The development of new tools for assessing the health of cultured shellfish larvae is crucial for aquaculture industries to develop and refine hatchery methodologies. We established a large-volume ecotoxicology/health stressor trial, exposing mussel (Perna canaliculus) embryos to copper in the presence of ethylenediaminetetraacetic acid (EDTA). GC/MS-based metabolomics was applied to identify potential biomarkers for monitoring embryonic/larval health and to characterise mechanisms of metal toxicity. Cellular viability, developmental abnormalities, larval behaviour, mortality, and a targeted analysis of proteins involved in the regulation of reactive oxygen species were simultaneously evaluated to provide a complementary framework for interpretative purposes and authenticate the metabolomics data. Trace metal analysis and speciation modelling verified EDTA as an effective copper chelator. Toxicity thresholds for P. canaliculus were low, with 10% developmental abnormalities in D-stage larvae being recorded upon exposure to 1.10 μg·L-1 bioavailable copper for 66 h. Sublethal levels of bioavailable copper (0.04 and 1.10 μg·L-1) caused coordinated fluctuations in metabolite profiles, which were dependent on development stage, treatment level, and exposure duration. Larvae appeared to successfully employ various mechanisms involving the biosynthesis of antioxidants and a restructuring of energy-related metabolism to alleviate the toxic effects of copper on cells and developing tissues. These results suggest that regulation of trace metal-induced toxicity is tightly linked with metabolism during the early ontogenic development of marine mussels. Lethal-level bioavailable copper (50.3 μg·L-1) caused severe metabolic dysregulation after 3 h of exposure, which worsened with time, substantially delayed embryonic development, induced critical oxidative damage, initiated the apoptotic pathway, and resulted in cell/organism death shortly after 18 h of exposure. Metabolite profiling is a useful approach to (1) assess the health status of marine invertebrate embryos and larvae, (2) detect early warning biomarkers for trace metal contamination, and (3) identify novel regulatory mechanisms of copper-induced toxicity.

Oxidative stress and redox imbalance adversely affect embryonic development. We developed two oxidative balance scores (OBS) that include dietary and nondietary exposures. We hypothesized that higher scores (i.e., lower oxidative stress) would be associated with lower risk of neural tube defects, orofacial clefts, conotruncal heart defects, and limb deficiencies. We used data from the National Birth Defects Prevention Study to create a dietary OBS based on intake of 13 nutrients and an overall OBS that included the 13 nutrients and eight additional nondietary factors related to oxidative balance (e.g., smoking). We used logistic regression to examine odds ratios associated with having low or high scores (i.e., <10th or >90th percentiles). Continuous models indicated reduced odds associated with high versus low scores (i.e., comparing odds at the 90th versus 10th percentile values of the distribution) on the overall OBS for cleft lip with or without cleft palate [adjusted odds ratio (aOR) 0.72, 95% confidence interval (CI) 0.63-0.82], longitudinal limb deficiency (aOR 0.73, CI 0.54-0.99), and transverse limb deficiency (aOR 0.74, CI 0.58-0.95); increased odds for anencephaly (aOR 1.40, CI 1.07-1.84); and primarily nonsignificant associations with conotruncal heart defects. Results for the dietary OBS were similar. This study provides some evidence that oxidative stress contributes to congenital anomalies related to neural crest cell development.

Oxidative stress (OS) and inflammation arising from cellular derangements at the fetal membrane-decidual interface (feto-maternal interface [FMi]) is a major antecedent to preterm birth (PTB). However, it is impractical to study OS-associated FMi disease state during human pregnancy, and thus it is difficult to develop strategies to reduce the incidences of PTB. A microfluidic organ-on-chip model (FMi-OOC) that mimics the in vivo structure and functions of FMi in vitro was developed to address this challenge. The FMi-OOC contained fetal (amnion epithelial, mesenchymal, and chorion) and maternal (decidua) cells cultured in four compartments interconnected by arrays of microchannels to allow independent but interconnected co-cultivation. Using this model, we tested the effects of OS and inflammation on both fetal (fetal → maternal) and maternal (maternal → fetal) sides of the FMi and determined their differential impact on PTB-associated pathways. OS was induced using cigarette smoke extract (CSE) exposure. The impacts of OS were assessed by measuring cell viability, disruption of immune homeostasis, epithelial-to-mesenchymal transition (EMT), development of senescence, and inflammation. CSE propagated (LC/MS-MS analysis for nicotine) over a 72-hour period from the maternal to fetal side, or vice versa. However, they caused two distinct pathological effects on the maternal and fetal cells. Specifically, fetal OS induced cellular pathologies and inflammation, whereas maternal OS caused immune intolerance. The pronounced impact produced by the fetus supports the hypothesis that fetal inflammatory response is a mechanistic trigger for parturition. The FMi disease-associated changes identified in the FMi-OOC suggest the unique capability of this in vitro model in testing in utero conditions.

Diafenthiuron, a broad-spectrum insecticide and acaricide used for agricultural crop protection, is highly toxic to nontarget organisms. However, the developmental toxicity of diafenthiuron and its underlying mechanisms are not fully understood. Thus, the purpose of this study was to investigate the developmental toxicity of diafenthiuron in zebrafish. Zebrafish embryos were exposed to diafenthiuron at different concentrations (0.01, 0.1, and 1 μM) from 3 to 120 h post fertilization (hpf). Diafenthiuron exposure significantly shortened the body lengths of zebrafish larvae and significantly decreased superoxide dismutase activity. It also downregulated the spatiotemporal expression of pomc and prl, marker genes involved in pituitary development. Moreover, diafenthiuron exposure downregulated the spatiotemporal expression of liver-specific marker, fabp10a, and inhibited the development of the liver, a detoxification organ. In conclusion, our data provide evidence of the developmental toxicity and hepatotoxicity of diafenthiuron in aquatic organisms, and they are instrumental for further environmental risk assessment of diafenthiuron in aquatic ecosystems.

6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored whether 6-gingerol can be used to improve the quality of in vitro-cultured porcine embryos. The results showed that 5 μM 6-gingerol significantly increased the blastocyst formation rates of porcine early embryos. 6-Gingerol attenuated intracellular reactive oxygen species accumulation and autophagy, increased intracellular glutathione levels, and increased mitochondrial activity. In addition, 6-gingerol upregulated NANOG, SRY-box transcription factor 2, cytochrome c oxidase subunit II, mechanistic target of rapamycin kinase, and RPTOR independent companion of MTOR complex 2 while downregulating Caspase 3, baculoviral IAP repeat containing 5, autophagy related 12, and Beclin 1. Most importantly, 6-gingerol significantly increased the levels of p-extracellular regulated protein kinase 1/2 while reducing the levels of p-c-Jun N-terminal kinase 1/2/3 and p-p38. These results indicate that 6-gingerol can promote the development of porcine early embryos in vitro.

Moringa stenopetala leaves (Baker f.) Cufod. (Moringaceae) are used as a staple food and traditional medicine for treating various diseases like malaria, hypertension, stomach pain, diabetes, elevated cholesterol, and removing the retained placenta. Its prenatal toxicity study is minimal. Thus, this study aimed to assess the toxic effects of a 70% ethanol extract of Moringa stenopetala leaf on the fetuses and placentas of pregnant Wistar rats.

Fresh leaves of Moringa stenopetala were collected, dried at room temperature, ground to powder, and extracted using 70% ethanol. For this study, five groups of animals, each containing ten pregnant rats, were used. Groups I-III were experimental groups and treated with 250, 500, and 1000 mg/kg body weight of Moringa stenopetala leaf extract, respectively. Groups IV and V were pair-fed and ad libitum control groups. The extract was given during gestation days 6 to 12. The fetuses were recovered at day 20 of gestation and examined for the presence of developmental delays, gross external malformations, skeletal and visceral defects. Gross and histopathological changes in the placenta were also evaluated.

Compared to the pair-fed control group, maternal daily food intake and weight gain were reduced in the 1000 mg/kg-treated group during the treatment and post-treatment periods. A significantly higher number of fetal resorptions was also seen in the 1000 mg/kg treatment group. The crown-rump length and fetal and placental weights were all significantly reduced in pregnant rats given 1000 mg/kg. However, there were no visible malformations in the visceral organs as well as external genitalia in all the treatment and control groups. About 40.7% of the fetuses in the 1000 mg/kg treated rats had no proximal hindlimb phalanges. In addition, light microscopic investigations of the placenta in the high-dose treated rats revealed structural changes in the decidual basalis, trophoblastic zone, and labyrinthine zones.

In conclusion, consumption of M. stenopetala leaves at a higher dose may have toxic effects on the development of rat fetuses. At a higher dose, the plant extract increased the number of fetal resorptions, reduced the number of fetuses, decreased the fetal and placental weights, and alter the placental histopathology. Thus, it is recommended to limit the excess feeding of M. stenopetala leaves during gestation.

To better define appropriate applications of our 3-dimensional testicular co-culture as a model for reproductive toxicology, we evaluated the ability of the model to capture structural and functional elements that can be targeted by reproductive toxicants. Testicular co-cultures were prepared from postnatal day 5 male rats and cultured with a Matrigel overlay. Following a 2-day acclimation period, we characterized functional pathway dynamics by evaluating morphology, protein expression, testosterone concentrations, and global gene expression at a range of timepoints from experimental days 0 to 21. Western blotting confirmed expression of Sertoli cell, Leydig cell, and spermatogonial cell-specific protein markers. Testosterone detected in cell culture media indicates active testosterone production. Quantitative pathway analysis identified Gene Ontology biological processes enriched among genes significantly changing over the course of 21 days. Processes enriched among genes significantly increasing through time include general developmental processes (morphogenesis, tissue remodeling, etc.), steroid regulation, Sertoli cell development, immune response, and stress and apoptosis. Processes enriched among genes significantly decreasing over time include several related to male reproductive development (seminiferous tubule development, male gonad development, Leydig cell differentiation, Sertoli cell differentiation), all of which appear to peak in expression between days 1 and 5 before decreasing at later timepoints. This analysis provides a temporal roadmap for specific biological process of interest for reproductive toxicology in the model and anchors the model to sensitive phases of in vivo development, helping to define the relevance of the model for in vivo processes.

Oxidative stress, which is a persistent state of elevated reactive oxygen species (ROS), is implicated in the pathogeneses of several diseases, making antioxidant-based therapeutics the aptest intervention. Nevertheless, the clinical failure of conventional low-molecular-weight (LMW) antioxidants in oxidative stress-related diseases to yield favorable therapeutic outcomes and an increased mortality rate attributable to their poor pharmacokinetic characteristics, necessitates the development of alternative therapeutics. In light of this, we designed and synthesized a new amphiphilic polymer functionalized with a clinically safe base polymer of poly(styrene-co-maleic anhydride) copolymer conjugated with the LMW pleiotropic antioxidant TEMPO (a potent antioxidant) and biocompatible poly(ethylene glycol) (TEMPO-installed PSMA-g-PEG), which self-assembles into nano-sized micelles (SMAPo^TN) under physiological conditions. We investigated its safety and antioxidant ability using zebrafish models. Common LMW antioxidants, such as 4-hydroxy-TEMPO (TEMPOL), vitamin C, N-acetyl-L-cysteine, and edaravone exposure induced phenotypic distortions, a manifestation of developmental toxicity, and resulted in high lethality in zebrafish larvae. LMW TEMPOL also adversely affected embryo hatchability, induced arrhythmia and cardiac edema, and failed to protect against oxidative stress. In contrast, exposure of zebrafish embryos to SMAPo^TN increased the hatchability, protected embryos against various inducers of oxidative stress, and did not induce any phenotypic alterations or discernible toxicity. Taken together, we conclude that SMAPo^TN surpasses LMW TEMPOL in terms of the ability to protect zebrafish, attributable to efficient ROS scavenging without perturbing normal redox homeostasis. These results imply that SMAPo^TN can be used as a therapeutic intervention against various oxidative stress-induced diseases. STATEMENT OF SIGNIFICANCE: Failure of low molecular weight (LMW) antioxidants to improve therapeutic index in various oxidative stress-related pathogenesis, attributable to their poor pharmacokinetic characteristics, greatly limits their clinical translation. To overcome this limitation, we developed a self-assembling antioxidant nanoparticle (SMAPo^TN) comprised of amphiphilic polymer; poly(styrene-co-maleic anhydride) conjugated with TEMPO as an antioxidant and biocompatible poly(ethylene glycol). Preliminary studies carried out in the in vivo models of zebrafish embryos confirmed that exposure of LMW antioxidant resulted in acute developmental toxicity, high lethality, and failure to rescue embryos against oxidative stress inducers. In contrast, SMAPo^TN did not exert discernible toxicity and significantly improved their survival under oxidative stress. Our finding establishes antioxidant nanoparticles as more suitable therapeutic intervention for oxidative stress-induced diseases than LMW antioxidants.