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Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Neutrophil extracellular traps (NETs) are a network structure composed of loose chromatin and embedded with multiple proteins. Here, we observed increased NETs deposition in the glomeruli of DKD patients and diabetic mice (streptozotocin-induced or db/db mice). After NETs were degraded with DNase I, diabetic mice exhibited attenuated glomerulopathy and glomerular endothelial cells (GECs) injury. We also observed alleviated glomerulopathy and GECs injury in peptidylarginine deiminase 4–knockout mice with streptozotocin-induced diabetes. In vitro, NETs-induced GECs pyroptosis was characterized by pore formation in the cell membrane, dysregulation of multiple genes involved in cell membrane function, and increased expression of pyroptosis-related proteins. Strengthening the GECs surface charge by oleylamine significantly inhibited NETs-induced GECs pyroptosis. These findings suggest that the GECs charge-related pyroptosis is involved in DKD progression, which is promoted by NETs.

Pre-eclampsia (PE) is deemed an ischemia-induced metabolic disorder of the placenta due to defective invasion of trophoblasts during placentation; thus, the driving role of metabolism in PE pathogenesis is largely ignored. Since trophoblasts undergo substantial glycolysis, this study aimed to investigate its function and regulatory mechanism by AMPK in PE development. Metabolomics analysis of PE placentas was performed by gas chromatography-mass spectrometry (GC-MS). Trophoblast-specific AMPKα1-deficient mouse placentas were generated to assess morphology. A mouse PE model was established by Reduced Uterine Perfusion Pressure, and placental AMPK was modulated by nanoparticle-delivered A769662. Trophoblast glucose uptake was measured by 2-NBDG and 2-deoxy-d-[

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Neutrophil extracellular traps (NETs) is a network structure composed of loose chromatin and embedded with multiple proteins. Here, we observed increased NETs deposition in the glomeruli of DKD patients and diabetic mice (streptozotocin-induced or db/db mice). After degrading NETs with DNase I, diabetic mice exhibited attenuated glomerulopathy and glomerular endothelial cells (GECs) injury. We also observed alleviated glomerulopathy and GECs injury in peptidylarginine deiminase 4 (PAD4)-knockout mice with streptozotocin-induced diabetes. In vitro, NET-induced GECs pyroptosis was characterized by pore formation in the cell membrane, dysregulation of multiple genes involved in cell membrane function, and increased expression of pyroptosis-related proteins. Strengthening the GECs surface charge by oleylamine significantly inhibited NETs-induced GECs pyroptosis. These findings suggest that the GECs charge related pyroptosis is involved in DKD progression which promoted by NETs.

Sufficient decidualization is necessary to maintain successful pregnancy. The physiological function and underlying molecular mechanisms of intergenic long non-coding RNA (lincRNAs) in this process remain largely unknown. Herein, we identified a lincRNA, RP24-315D19.10, which is highly expressed during mouse decidualization during early pregnancy, by performing RNA-sequencing (RNA-seq) analysis and weighted gene co-expression network analysis (WGCNA). Detailed cell and molecular assays revealed that lincRNA RP24-315D19.10 knockdown blocked decidualization in primary mouse endometrial stromal cells (mESCs), suggesting that RP24-315D19.10 is a promoting factor for decidualization. Mechanistically, cytoplasmic RP24-315D19.10 directly interacted with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) and upregulated its protein level. Moreover, we found that hnRNPA2B1 is involved in the regulation of decidualization through loss- and gain-of-function studiesin vitro. Clinically, patients diagnosed with spontaneous miscarriage were found to have lower hnRNPA2B1 levels than healthy individuals, suggesting that RP24-315D19.10-regulated hnRNPA2B1 may participate in the development and progression of early spontaneous abortion. Our study indicates that RP24-315D19.10 enhances endometrial decidualization in a hnRNPA2B1-dependent manner, providing further insights into this physiological process.

Existing evidence suggests that adverse pregnancy outcomes are closely related to dietary factors. Folate plays an important role in neural tube formation and fetal growth, folate deficiency is a major risk factor of birth defects. Our early studies showed that folate deficiency could impair enddecidualization, however, the mechanism is still unclear. Dysfunctional autophagy is associated with many diseases. Here, we aimed to evaluate the adverse effect of folate deficiency on endometrial decidualization, with a particular focus on endometrial cell autophagy. Mice were fed with no folate diet in vivo and the mouse endometrial stromal cell was cultured in a folate-free medium in vitro. The decrease of the number of endometrial autophagosomes and the protein expressions of autophagy in the folate-deficient group indicated that autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation were inhibited. Autophagic flux examination using mCherry-GFP-LC3 transfection showed that the fusion of autophagosomes with lysosomes was inhibited by folate deficiency. Autophagy inducer rapamycin could reverse the impairment of folate deficiency on endometrial decidualization. Moreover, folate deficiency could reduce autophagy by disrupting AMPK/mTOR signaling, resulting in aberrant endometrial decidualization and adverse pregnancy outcomes. Further co-immunoprecipitation examination showed that decidual marker protein Hoxa10 could interact with autophagic marker protein Cathepsin L, and the interaction was notably reduced by folate deficiency. In conclusion, AMPK/mTOR downregulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice, which could result in adverse pregnancy outcomes. This provided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency.

Decidualization of endometrial stromal cells (ESCs) accompanied with embryo implantation is a key process in mammalian reproduction. Evidence suggests that maintenance of decidual cells function is essential. As a critical part in post-transcriptional gene regulation, microRNAs (miRNAs/miR) have been confirmed to be involved in decidualization. However, whether microRNAs regulate decidual cells function has not been reported. Aiming to clarify the role and potential mechanism of miRNAs in decidual cells, artificial induced decidualization model in mice was established. There are 94 differentially expressed miRNAs (≥two-fold change) between decidualized and non-decidualized tissues, including 60 upregulated and 34 downregulated miRNAs. Of the differentially expressed miRNAs, mmu-miR-21a is up-regulated. RT-qPCR also confirmed the up-regulation of mmu-miR-21a following decidualization in vivo and in vitro, and bioinformatic analysis and luciferase activity assay revealed Pdcd4 to be the target gene of mmu-miR-21a. Inhibition of mmu-miR-21a restrained secretory function of decidual cells induced by mESCs, accompanied with increase of Pdcd4 expression and resulted in the increase of cell apoptosis. In addition, we also determined the expression of hsa-miR-21 and Pdcd4 in human proliferative endometrial tissues and decidua tissues. hsa-miR-21 showed higher expression in human decidua tissues compared with proliferative endometrial tissues, while expression of Pdcd4 was contrary to that of hsa-miR-21. Similarly, cell apoptosis increased significantly in human endometrial stromal cell line in response to inhibition of hsa-miR-21. Collectively, we conclude that mmu-miR-21a/hsa-miR-21 may play a key role in regulating the function of decidual cells by inhibiting cell apoptosis through targeting Pdcd4.

Background: Recent studies have demonstrated that endometrial DNA methylation is essential for embryo implantation during early pregnancy. Dnmt3a is one of the key enzymes for DNA methylation and could be expressed in the endometrium regularly at this stage. Objective and Methods: In this study, we conditionally ablated uterine Dnmt3a using progesterone receptor-cre (Pgrcre) to define the physiological roles of Dnmt3a in female reproduction. Results: We found that ovarian function was not apparently altered and the number of embryo implantation sites in Dnmt3aloxP/loxP Pgrcre/+ (cKO) was not significantly varied during early pregnancy. Western blotting and immunohistochemistry results showed no difference in expression or location of the estrogen receptor α (ERα) and mucin 1 (Muc1), the marker of uterine receptivity. Although the expression of decidual markers, matrix metalloproteinase-2 (Mmp2), matrix metalloproteinase-9(Mmp9), and bone morphogenetic protein-2 (Bmp2), was slightly decreased in Dnmt3a cKO females, the gross morphology of mice uteri during decidualization was not significantly influenced. In the artificial induction of the decidualization model, there was also no remarkable difference in visually observed morphology or uterine weight in Dnmt3a cKO. Lastly, a continuous breeding study showed that the fertility of Dnmt3a cKO female mice was not strikingly altered. Conclusion: Overall, these results demonstrated that although some decidual markers are expressed abnormally, conditional knockout of Dnmt3a in the uterus did not significantly affect the endometrial function during embryo implantation; the embryo could implant into the endometrium normally.

Benzo(a)pyrene (BaP) is a well-known environmental endocrine pollutant, which has ovarian toxicity in mammals. Ovarian corpus luteum (CL), as the main source of progesterone synthesis in early pregnant female, requires a large number of mitochondria for energy supply. We previously demonstrated that BaP and its metabolite benzo(a)pyren-7, 8-dihydrodiol-9, 10-epoxide (BPDE) inhibited the ovarian melatonin receptors (MTRs) expression and decreased the levels of estrogen and progesterone during early pregnancy in mice. Emerging researches show that MTRs also exist on mitochondrial membrane and participate in the regulation of mitochondrial function. However, the relationship between BaP, MTRs on mitochondrial membrane and mitochondrial function remains unknown. Consequently, this study focuses on the effect and potential mechanism of BaP on ovarian luteal mitochondrial function during early pregnancy. We found that BaP and its metabolite BPDE decreased MTRs in early pregnant CL and luteinized KGN cells, especially in mitochondria. Furthermore, BaP or BPDE up-regulated the expression of SIRT3, Mfn2 and Drp-1, damaged mitochondrial morphology and decreased the MMP and the ATP levels, thereby causing mitochondrial dysfunction. Notably, activation of the MTRs on mitochondrial membrane by MTRs agonist ramelteon partially alleviated BPDE-induced up-regulation of SIRT3, Mfn2 and Drp-1, reduced mitochondrial fragmentation and enhanced the MMP and the ATP levels, thus restoring the expression of steroid rate-limiting enzymes. Together, these findings firstly proved that BaP and BPDE down-regulate MTRs on mitochondrial membrane, and further injure mitochondrial function in early pregnant rats' CL, which provides a new insight for understanding the exact mechanism of the BaP-induced ovarian toxicity.

Circular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs that play important roles in gene expression regulation. This study aimed to evaluate the potential role of circRNAs in decidual tissue of patients with early recurrent miscarriage (RM). We constructed circRNA expression profiles in decidual tissue using microarray data. A total of 123 differentially expressed circRNAs, including 78 upregulated and 45 downregulated circRNAs were detected in the early RM group compared with the control group (P

Fetal growth restriction (FGR) is a condition in which a newborn fails to achieve his or her prospective hereditary growth potential. This condition is associated with high newborn mortality, second only to that associated with premature birth. FGR is associated with maternal, fetal, and placental abnormalities. Although the placenta is considered to be an important organ for supplying nutrition for fetal growth, research on FGR is limited, and treatment through the placenta remains challenging, as neither proper uterine intervention nor its pathogenesis have been fully elucidated. Yes-associated protein (YAP), as the effector of the Hippo pathway, is widely known to regulate organ growth and cancer development. Therefore, the correlation of the placenta and YAP was investigated to elucidate the pathogenic mechanism of FGR. Placental samples from humans and mice were collected for histological and biomechanical analysis. After investigating the location and role of YAP in the placenta by immunohistochemistry, we observed that YAP and cytokeratin 7 have corresponding locations in human and mouse placentas. Moreover, phosphorylated YAP (p-YAP) was upregulated in FGR and gradually increased as gestational age increased during pregnancy. Cell function experiments and mRNA-Seq demonstrated impaired YAP activity mediated by extracellular signal-regulated kinase inhibition. Established FGR-like mice also recapitulated a number of the features of human FGR. The results of this study may help to elucidate the association of FGR development with YAP and provide an intrauterine target that may be helpful in alleviating placental dysfunction.Summary SentenceImpaired hippo pathway in placenta might be a causative factor of fetal growth restriction.

Embryo implantation is essential for normal pregnancy. Decidualization is known to facilitate embryo implantation and maintain pregnancy. Uterine stromal cells undergo transformation into decidual cells after embryo attachment to the endometrium. Pyruvate kinase M2 (PKM2) is a rate limiting enzyme in the glycolysis process which catalyzes phosphoenolpyruvic acid into pyruvate. However, little is known regarding the role of PKM2 during endometrial decidualization. In this study, PKM2 was found to be mainly located in the uterine glandular epithelium and luminal epithelium on day 1 and day 4 of pregnancy and strongly expressed in the decidual zone after embryo implantation. PKM2 was dramatically increased with the onset of decidualization. Upon further exploration, PKM2 was found to be more highly expressed at the implantation sites than at the inter-implantation sites on days 5 to 7 of pregnancy. PKM2 expression was also significantly increased after artificial decidualization both in vivo and in vitro. After PKM2 expression was knocked down by siRNA, the number of embryo implantation sites in mice on day 7 of pregnancy was significantly reduced, and the decidualization markers BMP2 and Hoxa10 were also obviously downregulated in vivo and in vitro. Downregulated PKM2 could also compromise cell proliferation in primary endometrial stromal cells and in Ishikawa cells. The migration rate of Ishikawa cells was also obviously suppressed by si-PKM2 according to the wound healing assay. In conclusion, PKM2 might play an important role in decidualization during early pregnancy, and cell proliferation might be one pathway for PKM2 regulated decidualization.

Objective: To investigate whether serum bisphenol A (BPA) concentration is related to the occurrence of dyslipidemia. Methods: A total of 574 adults were enrolled at baseline and followed up for 5 years. Concentrations of serum BPA, triglycerides (TGs), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were measured. Dyslipidemia was defined as the existence of one or more of the following conditions: high-LDL-cholesterolemia (LDL ≥140 mg/dL), hypertriglyceridemia (TGs ≥150 mg/dL), or low-HDL-cholesterolemia (HDL

Previous research on the role of insulin has focused on metabolism. This study investigated the effect of insulin on angiogenesis in endometrial decidualization. High insulin-treated mouse model was constructed by subcutaneous injection of insulin. Venous blood glucose, serum insulin, P4, E2, FSH and LH levels in the pregnant mice were detected by ELISA. Decidual markers, angiogenesis factors and decidual vascular network were detected during decidualization in the pregnant mouse model and an artificially induced decidualization mouse model. Tube formation ability and angiogenesis factors expression were also detected in high insulin-treated HUVECS cells. To confirm whether autophagy participates in hyperinsulinemia-impaired decidual angiogenesis, autophagy was detected in vivo and in vitro. During decidualization, in the condition of high insulin, serum insulin and blood glucose were significantly higher, while ovarian steroid hormones were also disordered (P P in vivo and in vitro (P P P

The increasing use of cerium dioxide nanoparticles (CeOHere, we constructed a mouse exposure model with different doses of CeOMaternal exposure to CeO

Uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. Recent advances in molecular technologies have allowed the unprecedented mapping of epigenetic modifications during embryo implantation. DNA methyltransferase 3a (DNMT3A) and DNMT3B are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. It was reported that conditional knockout of Dnmt3a in the uterus does not markedly affect endometrial function during embryo implantation, but the tissue-specific functions of Dnmt3b in the endometrium during embryo implantation remain poorly understood to investigate the role of Dnmt3b during peri-implantation period. Here, we generated Dnmt3b conditional knockout (Dnmt3bd/d) female mice using progesterone receptor-Cre mice and examined the role of Dnmt3b during embryo implantation. Dnmt3bd/d female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. Furthermore, results showed loss of Dnmt3b did not lead to altered genomic methylation patterns of the decidual endometrium during early pregnancy. Transcriptome sequencing analysis of uteri from day 6 pregnant mice identified phosphoglycerate kinase 1 (Pgk1) as one of the most variable genes in Dnmt3bd/d decidual endometrium. Potential roles of PGK1 in the decidualization process during early pregnancy were confirmed. Lastly, the compromised decidualization upon the downregulation of Dnmt3b could be reversed by overexpression of Pgk1. Collectively, our findings indicate that uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects.

Exposure to benzo (a)pyrene (BaP) has been confirmed to interfere with embryo implantation. As the primary organ of progesterone synthesis during early pregnancy, the ovarian corpus luteum (CL) is essential for embryo implantation and pregnancy maintenance. We previously demonstrated that BaP impaired luteal function, but the molecular mechanism remains unclear. In CL cells, mitochondria are the main sites of progesterone synthesis. Mitophagy, a particular type of autophagy, regulates mitochondrial quality by degrading damaged mitochondria and ensuring the homeostasis of cell physiology. Therefore, the present study investigated the effects and the potential molecular mechanisms of BaP on ovarian mitophagy during early pregnancy. We found that BaP and its metabolite, BPDE, inhibited autophagy and PINK1/Parkin-mediated mitophagy in the pregnant ovaries and luteinized granulosa cell, KGN. Notably, adenine nucleotide translocator 1 (ANT1), a crucial mediator of PINK1-dependent mitophagy, was suppressed by BaP and BPDE both in vivo and in vitro. The inhibition of ANT1 leads to the decrease in the PINK1 bound to the outer membrane of mitochondria and consequently reduces recruitment of Parkin to the mitochondria, which is required for the subsequent clearance of mitochondria. Meanwhile, exposure to BPDE also damaged mitochondrial function, causing the reduction in mitochondrial potential and ATP production. Overexpression of ANT1 in KGN cells partially relieved the inhibition of mitophagy caused by BPDE, restored mitochondrial function and expression of hormone synthesis-associated genes. Collectively, our study firstly clarified that BaP and BPDE suppress mitophagy of CL cells via the ANT1-PINK1-Parkin pathway, which provides a new insight to explore the detailed mechanism of the BaP-induced ovarian toxicity.

Advanced maternal age (AMA) pregnancies are rapidly increasing and are associated with aberrant trophoblast cell function, poor placentation, and unfavorable pregnancy outcomes, presumably due to premature placental senescence. SIRT1 is an NAD+‐dependent deacetylase with well‐known antiaging effects, but its connection with placental senescence is unreported. In this study, human term placentas and first‐trimester villi were collected from AMA and normal pregnancies, and a mouse AMA model was established by cross breeding young and aged male and female C57 mice. SIRT1 expression and activity in HTR8/SVneo cells were genetically or pharmacologically manipulated. Trophoblast‐specific Sirt1‐knockout (KO) mouse placentas were generated by mating Elf5‐Cre and Sirt1 fl/fl mice. Trophoblast cell mobility was assessed with transwell invasion and wound‐healing assays. SIRT1‐binding proteins in HTR8/SVneo cells and human placental tissue were identified by mass spectrometry. We identified SIRT1 as the only differentially expressed sirtuin between AMA and normal placentas. It is downregulated in AMA placentas early in the placental life cycle and is barely impacted by paternal age. SIRT1 loss upregulates P53 acetylation and P21 expression and impairs trophoblast invasion and migration. Sirt1‐KO mouse placentas exhibit senescence markers and morphological disruption, along with decreased fetal weight. In trophoblasts, SIRT1 interacts with vimentin, regulating its acetylation. In conclusion, SIRT1 promotes trophoblast epithelial−mesenchymal transition (EMT) to enhance invasiveness by modulating vimentin acetylation. AMA placentas are associated with premature senescence during placentation due to SIRT1 loss. Therefore, SIRT1 may be an antiaging therapeutic target for improving placental development and perinatal outcomes in AMA pregnancies., Both the first‐trimester villi and full‐term placentas from pregnancies of advanced maternal age (AMA) are associated with the significant downregulation of SIRT1 and accumulation of P53, P21, and β‐galactosidase in trophoblasts, where SIRT1 directly interacts with vimentin and modulates the acetylation status of vimentin, thus affecting the EMT process and ultimately influencing the invasiveness and mobility of trophoblasts. Both P53–P21 axis‐dependent cellular senescence and vimentin acetylation‐induced EMT suppression may orchestrate premature placental senescence and eventually lead to a variety of adverse pregnancy outcomes.

Bisphenol A (BPA) is a common endocrine disruptor and a high-fat diet (HFD) also affects fertility. However, little is known about the long-term consequences of simultaneous exposure to BPA and a HFD on reproductive health. Herein, we assessed the effects of maternal exposure to BPA in combination with a HFD on reproductive function in subsequent generations of female mice and evaluated its effects on the hypothalamic-pituitary-gonadal axis. We found that the combination of maternal exposure to BPA and a HFD led to increased urine BPA levels, precocious puberty, altered estrous cyclicity, decreased follicle numbers, and altered hypothalamic Kiss1 methylation status in F1 and F2 mice. Therefore, we demonstrated that maternal exposure to BPA in combination with a HFD exerts a trans-generational effect on female reproduction.

Ethylparaben (EtP) and propylparaben (PrP) are common preservatives and well-known endocrine-disrupting chemicals. Studies have demonstrated that they can reduce female fertility, but the underlying mechanism, especially that on embryo implantation, is still poorly understood. Endometrial decidualization is a critical event for embryo implantation. In this study, we aimed to explore the effects of EtP/PrP on endometrial decidualization. Pregnant mice were dosed daily by oral gavage with EtP at 0, 400, 800 and 1600 mg/kg or with PrP at 0, 625, 1250 and 2500 mg/kg from Day 1 of pregnancy until sacrifice. The results showed that the rate of pregnant mice with impaired embryo implantation, whose number of implantation sites was less than 7, was significantly increased after exposure to 1600 mg/kg EtP or 2500 mg/kg PrP. Further study found that the expression of endometrial decidualization markers HOXA10, MMP9 and PR was significantly downregulated in 1600 mg/kg EtP group and 2500 mg/kg PrP group. Notably, serum oestrogen and progesterone levels were significantly increased, whereas the expression of uterine oestrogen receptor and progesterone receptor was decreased following 1600 mg/kg EtP or 2500 mg/kg PrP exposure. In the breeding test, fewer offspring were found after females were exposed to 1600 mg/kg EtP or 2500 mg/kg PrP in early pregnancy. This demonstrated that exposure to EtP/PrP interfered with embryo implantation by compromising endometrial decidualization in early-stage pregnant mice. Disorders of reproductive hormones and hormone receptor signals could be responsible for impaired decidualization. This study broadened the understanding on the biological safety of EtP and PrP.

Background: Obesity is associated with many adverse effects on female fertility. Obese women are more likely to have ovulatory dysfunction due to dysregulation of the hypothalamic-pituitary-ovarian axis. However, ovarian function in obese women during early pregnancy still needs further assessment. Methods: Obesity was induced in C57BL6/J mice using high-fat diets (HFD) for 12 weeks; In vitro high-fat model was established with KGN cells treated with Oleate acid and Palmitic acid. Ovarian morphology of obese mice in early pregnancy was assessed by Hematoxylin and Eosin staining and its function was assessed using ELISA, Western blotting and Immunohistochemistry. The Oil Red O staining and Transmission electron microscopy were used to detect fatty acid accumulation and specific markers relating to ovarian functional mechanism were assessed by Real time PCR, Western blotting, Lactate detection, ATP detection, Biochemical analyses and ELISA.Results: The results of this study showed that during early pregnancy, the number of corpus luteum, serum estradiol and progesterone levels, and the expression of genes CYP19A1, CYP11A1 and StAR, which are related to steroid biosynthesis, were significantly increased in HFD female mice. HFD-fed mice also showed a significant increase in ovarian lipid accumulation on day 7 of pregnancy. Genes involved in fatty acid synthesis (Acsl4 and Elovl5) and fatty acid uptake and transport (Slc27a4), together with the β-oxidation rate-limiting enzyme (Cpt1a) were significantly upregulated in HFD-fed mice. Specifically, there was abnormal elevation of ATP level and aberrant expression of tricarboxylic acid cycle (TCA) and electron transport chain related genes in the ovary of HFD pregnant mice. Treatment of KGN cells with etomoxir targeting β-oxidation of fatty acid, showed decrease tricarboxylic acid cycle and electron transport chain. The elevated ATP level and the increased estradiol and progesterone levels were reversed. Conclusions: This study indicated that during early pregnancy, high-fat diet and induced-obesity increased fatty acid β-oxidation, which in turn increase the tricarboxylic acid cycle and the electron transport chain, and consequently increases ATP production and ovarian dysfunction.

Bisphenol-A (BPA) is a common environmental pollutant, and exposure to it is associated with proteinuria and may predict the progression of chronic kidney disease; however, the mechanism is not clear. Neutrophil extracellular traps (NETs) are a DNA skeleton coated with various proteases, and it is associated with various types of autoimmune nephritis. In this study, we examine whether NETs is involved in BPA-induced chronic kidney injury. In vivo, BPA exposure resulted in impaired renal function and altered renal morphology, including glomerular mesangial matrix expansion and increased renal interstitial fibroblast markers. Meanwhile, more dsDNA can be detected in the serum, and the NETs-associated proteins, MPO and citH3 were deposited in the renal system. In vitro, BPA and NETs treatment caused podocyte injury, a loss of marker proteins and disorder in the actin skeleton. After NETs inhibition via DNase administration, BPA-induced injuries were significantly relieved. In conclusion, the increase of NETosis in circulation and the renal system during BPA exposure suggests that NETs may be involved in BPA-induced chronic kidney injury.

We fabricate a novel electrochemical biosensor based on the specific thymine-Hg2+-thymine (T-Hg2+-T) base pair for the highly sensitive detection of mercury ions (Hg2+) and utilize toluidine blue (TB) as a redox indicator that is combined with a hybridization chain reaction (HCR) for signal amplification. The dandelion-like CuO (D-CuO) microspheres that were assembled using Au nanoparticles were first introduced as support materials, which produced more active sites for the thiolated probe (P1) combination. Then, the presence of Hg2+ induced P1 to hybridize with the other oligonucleotide (P2) through Hg2+-mediated T-Hg2+-T complexes. In addition, the partial sequence of P2 acted as an initiator sequence, which led the two hairpin DNA (H1 and H2) strands to collectively form the extended double-strand DNA through the HCR process on the electrode surface. TB was employed to interact with the double strands and produce an efficient electrochemical signal. The proposed strategy combined the amplification of the HCR and the inherent redox activity of TB and utilized D-CuO/Au composites, which exhibited high sensitivity for Hg2+ determination. Under the optimum conditions, the proposed biosensor showed a prominent response for Hg2+, including a linear range from 1 pM to 100 nM and a detection limit of 0.2 pM (S/N = 3). Moreover, the new biosensor proved its potential application for trace Hg2+ determination in environmental water samples.

Low molecular weight pollutants from foods have aroused global attention due to their toxicity after long-time exposure. There is an increased demand for appropriate methods to detect these pollutants in foods. In this study, a brand-new type of nano metal–organic coordination polymers (MOCPs) nanocarriers (3D sakura-shaped copper (II) ions@ L -glutamic acid ( L -Glu)) has been first synthesized. We herein demonstrate a facile chelated method that allows the combination of copper (II) ions and L -Glu. A series of controlled experiments have revealed that the reaction time and the ratio of reactants played the crucial roles in affecting the morphology of the final product. 3D sakura-shaped Cu@ L -Glu combined with palladium-platinum nanoparticle (Pd-PtNPs) to obtain Cu@ L -Glu/Pd-PtNPs acting as the signal tag, which applied in electrochemical aptasensor for ultrasensitive detection of zearalenone (ZEN). A glassy carbon electrode was first modified with spherical Au-PANI-Au nanohybrids to enhance the conductivity and immobilize more amino modified ZEN aptamer. Cu@ L -Glu/Pd-PtNPs were labeled with Complementary DNA (partial matching with ZEN aptamer) to form bioconjugates for signal amplification. After the hybridization reaction of ZEN aptamer and the bioconjugates, a significant electrochemical signal from the catalysis of H2O2 by Cu@ L -Glu/Pd-PtNPs can be observed. ZEN competed with bioconjugates for binding to ZEN aptamer, resulting in decreased the electrochemical signal. Chronoamperometry was applied to record the final electrochemical signals. Under optimal conditions, the electrochemical aptasensor exhibited desirable sensitive detection of ZEN with a wide linearity ranging from 1 fg/mL to 100 ng/mL and a relatively low detection limit of 0.45 fg/mL (S/N = 3). Furthermore, the proposed electrochemical aptasensor shows excellent selectivity to the ZEN in the presence of possible interfering substances, and has potential application for ZEN detection in food samples.

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg-1 ·day -1 BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.

Circular RNAs (circRNAs), a class of newly discovered endogenous noncoding RNAs, have been found to play important roles in regulating gene expression and participate in several biological processes. However, their specific roles in embryo implantation remain unclear. This study aims to investigate the roles of circRNAs in embryo implantation. In the current study, we screened the expression profiles of circRNAs between implantation sites and interimplantation sites in the endometrial tissue of early pregnant mice on Day 5 using microarray assay. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) of four dysregulated circRNAs was performed to validate the microarray results. Bioinformatics analyses, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and circRNA-microRNA (miRNA)-messenger RNA (mRNA) negative correlation network analyses were performed. The microarray results showed 101 upregulated and 75 downregulated circRNAs (fold change [FC] ≥ 1.5 and p value < 0.05) at implantation sites compared with interimplantation sites. Four randomly selected circRNAs were successfully verified by qRT-PCR. The GO and KEGG analyses revealed some meaningful terms. Moreover, based on circRNAs microarray data and the miRNA and mRNA high-throughput data previously published by our groups, differently expressed circRNAs, miRNAs, and mRNAs were performed to conjoint analysis. Then circRNA-miRNA-mRNA negative correlation networks were constructed and 21 downregulated circRNAs, 14 upregulated miRNAs, and 79 downregulated mRNAs were involved in the networks. The findings of this study may provide a novel perspective on circRNAs and lay a foundation for future research into the potential roles of circRNAs in embryo implantation.

Folate deficiency is a major risk factor of birth defects. Mechanistic studies on folate deficiency resulting in birth defects have mainly focused on fetal development. There have been few studies on folate deficiency from the point of view of the mother's uterus. In our previous study, we demonstrated that folate deficiency inhibits apoptosis of decidual cells, thereby restraining decidualization of the endometrium and impairing pregnancy. In this study, we further investigated the potential mechanism by which folate deficiency decreases endometrial apoptosis during decidualization. To investigate whether endometrium autophagy was inhibited under folate deficiency during decidualization, we performed real-time PCR for endometrial LC3 and P62 on day 6 (D6) to D8 of pregnancy in mice, and both were significantly changed compared to non-folate-deficient mice. Western blots showed that LC3-II and P62 were also changed in folate-deficient mice. Compared with control mice, a few punctuate LC3-II structures were detected in the folate deficiency group by immunofluorescence. Transmission electron micrographs of decidual cells on D8 showed that there were no evident autophagosomes in the folate deficiency group. In addition, apoptosis-related protein analysis by western blotting, TUNEL staining and flow cytometry showed that decreased endometrial apoptosis on D8 of pregnancy under folate deficiency was reversed after treatment with rapamycin, an autophagy inducer. ROS measurement showed that the endometrium ROS level was reduced by folate deficiency and that rapamycin reversed this effect on day 8 of pregnancy. All the results suggest that inhibiting endometrial autophagy may be implicated in the decreased endometrial apoptosis under folate deficiency during decidualization.

Exposure to ethephon (ETH), a plant growth regulator commonly used for several purposes, can potentially decrease sperm numbers and viability. Occasional findings regarding ETH effects on female reproduction during early pregnancy have also been reported. During early pregnancy, endometrial decidualization is a critical event for embryo implantation and pregnancy maintenance. Thus, we aimed to explore the effect and mechanism of ETH on endometrial decidualization both in vivo and in vitro. Mice were gavaged with 0 and 285 mg/kg b.w. ETH from gestational days (GD)1 until sacrifice, whereas pseudopregnant mice from pseudopregnant day 1 (PPD-1) until PPD-8. Primary mouse endometrial stromal cells (mESCs) received 640 ug/ml ETH and added E2 and P4 to induce decidualization. Results indicated female albino CD1 mice exposed to high dose of ETH (285 mg/kg b.w.) by oral gavage, the number of embryo implantation sites on GD6 and GD8 were significantly decreased, the levels of serum E2 and P4 on GD8 were significantly decreased. Compared with the control group, the decidualization response artificially induced by corn oil in pseudopregnant mice and by E2 and P4 in primary mouse endometrial stromal cells (mESCs) was weakened in the high dose of ETH treated group. The high dose, 285 mg/kg b.w ETH treated group altered the expression of endometrial decidual markers on GD6 and GD8. The triglyceride and fatty acid metabolism-related genes were significantly increased after female albino CD1 mice exposed to high does, 285 mg/kg b.w ETH on GD6 and GD8. GPR120 was substantially reduced after ETH treatment. When overexpression of GPR120, the compromised decidualization induced by ETH treatment was rescued. Furthermore, molecular docking presented Thr234 and His251 of GPR120 as preferred binding sites for ETH. Mutation of these two sites rescued the compromised decidualization induced by ETH. In conclusion, we demonstrated that ETH exposure could impair decidualization during early pregnancy. GPR120 expression and binding between GPR120 and ETH are crucial for impaired decidualization mediated via ETH.

Decidualization accompanies with extensive stromal cell proliferation and differentiation, is a crucial step in early pregnancy. Aberrant decidualization is linked to infertility and miscarriage but the mechanisms remain unclear. Carnitine palmitoyltransferase 1A (CPT1A) is an enzyme catalyzing key steps in the fatty acid beta-oxidation pathway. The objective of this study was to investigate the role of CPT1A in decidualization during early pregnancy. An increased expression of CPT1A was found both in Days 6 and 7 as compared with in Days 1, 4 and 5. Further examination showed that on days 5–7 of pregnancy, the protein level of CPT1A was strongly up-regulated at implantation sites compared with inter-implantation sites, the location of CPT1A protein was distributed in the decidual zone. Upon further exploration, CPT1A expression was significantly increased in response to artificially induced decidualization both in vivo and in vitro. After down-regulating CPT1A expression by CPT1A-small interfering RNA (siCPT1A) in primary mouse endometrial stromal cells, expressions of decidualization markers and cell proliferation markers were decreased. After siCPT1A was transfected into the mouse uterus, decidualization impaired and then led to the loss of the implanted embryos. Thus, CPT1A is important for decidualization in mice and it may regulate the stromal cell proliferation progress. It is worth noting that the expression of CPT1A protein of human decidua was significantly decreased in spontaneous abortion groups compared to normal pregnancy groups. Collectively, CPT1A is essential for endometrium of early pregnant mice and humans.

Butylated hydroxytoluene (BHT), one of the most widely used synthetic phenolic antioxidants, is a popular food additive. Previous studies have reported the possible health hazards of BHT. However, BHT effects on female reproduction, especially on endometrial decidualization, are still unknown. During early pregnancy, decidualization plays important roles for embryo implantation and pregnancy establishment. This study aimed to explore the effects of BHT on endometrial decidualization in pregnant mice. The pregnant mice received BHT via intraperitoneal injection at doses of 0, 200, and 400 mg/kg/day from day 1 (D1) of pregnancy until sacrifice. Under BHT exposure, maternal body weight was significantly decreased during early pregnancy. Compared with the control group, the number of implantation sites and uterine weight were significantly reduced in the BHT groups. The uterine lumen failed to close after BHT exposure, and the decidual morphology of endometrial stromal cells was inhibited by BHT. Furthermore, BHT significantly decreased the expression of endometrial decidual markers including COX2, HOXA10, and MMP9. Notably, the levels of serum estrogen (E2) and progesterone (P4) and expression levels of uterus estrogen receptor α (ERα) and progesterone receptor (PR) during early pregnancy were significantly upregulated following BHT exposure. In conclusion, these results demonstrated that gestational BHT exposure could inhibit decidualization of mouse endometrium during early pregnancy. The disorders of reproductive hormones and changes of hormone receptor signals could be responsible for the impaired decidualization. This study provided new evidence for the deleterious effects of BHT on female reproduction and revealed the potential reproductive toxicity of synthetic phenolic antioxidants.

Benzo(a)pyrene (B(a)P) is a widespread persistent organic pollutant (POP) and a well-known endocrine disruptor. Exposure to BaP is known to disrupt the steroid balance and impair embryo implantation, but the mechanism under it remains unclear. The corpus luteum (CL), the primary source of progesterone during early pregnancy, plays a pivotal role in embryo implantation and pregnancy maintenance. The inappropriate luteal function may result in implantation failure and spontaneous abortions. Therefore, this study was conducted to assess the effects and potential mechanisms of B(a)P on the CL function. Our results showed that pregnant mice received B(a)P displayed impaired embryo implantation and dysfunction of ovarian CL. The estrogen and progesterone levels decreased by B(a)P. In vitro, exposure to BPDE, which is the metabolite of B(a)P, affected the luteinization of granular cell KK-1. Additionally, melatonin and its receptors, which are important for ovarian function and anti-oxidative damage, were affected by B(a)P or BPDE. B(a)P or BPDE-treated alone impaired antioxidant capacity of ovarian granulosa cells, caused an increasing of ROS and cell apoptosis, and disrupted the PI3K/AKT/GSK3β signaling pathway in vivo and in vitro. Co-treatment with melatonin alleviated B(a)P or BPDE-induced CL dysfunction by ameliorating oxidative stress, counteracting phosphorylation of PI3K/AKT/GSK3β signaling pathway, decreasing the apoptosis of the ovarian cells. Moreover, activation of the melatonin receptor by ramelteon in KK-1 cells exhibits an analogous protective effect as melatonin. In conclusion, our findings not only firstly clarify the potential mechanisms of BaP-induced CL dysfunction, but also extend the understanding about the ovarian protection of melatonin and its receptors against B(a)P exposure.

Supplementary Methods: 1 Mouse endometrial stromal cell isolation and culture; 2. Immunofluorescence cytology. 2. Supplementary Figures: Figure S1. The serum folate concentration was detected by electro-chemiluminescence immunoassay; Figure S2. Cell indentification was induced by hormone treatment in primary mESCs (in vitro); Figure S3. The key mRNA expressions of autophagy by qRT-PCR in primary mESCs (in vitro). Supplementary Tables: Table S1. Antibodies were used in western blot; Table S2. Sequences of forward and reverse primers were used in RT-PCR (5’→3’).