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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.

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.

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.

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

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.

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.

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.

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.

Previous studies have investigated the lower embryo implantation rates in women with polycystic ovary syndrome, obesity and type 2 diabetes, and specifically the association between the abnormal oocyte and embryo and hyperinsulinemia. The importance of hyperinsulinemia on maternal endometrium receptivity remains to be elucidated. The present study used a hyperinsulinemic mouse model to determine whether hyperinsulinemia may affect endometrial receptivity. An insulin intervention mouse model was first established. The serum levels of insulin, progesterone and estradiol were subsequently detected by ELISA assay analysis. The number of implantation sites was recorded using Trypan blue dye and the morphology of mice uteri was investigated using hematoxylin and eosin staining. The expression levels of molecular markers associated with endometrial receptivity were detected by reverse transcription‑quantitative polymerase chain reaction, western blotting and immunohistochemistry analyses. Finally, the importance of mechanistic target of rapamycin (mTOR) expression following insulin treatment was determined. Mice treated with insulin developed insulin resistance and hyperinsulinemia. The number of implantation sites following insulin treatment did not differ between the control and insulin‑treated groups. Additionally, no significant morphological alterations in mice uteri between control and insulin‑treated groups were observed. However, the expression levels of estrogen receptor (Esr) 1, Esr2, progesterone receptor and homeobox A10 associated with endometrial receptivity, were imbalanced during endometrium receptivity when maternal hyperinsulinemia was induced. Western blot analysis revealed that expression levels of endometrial phosphorylated (p)‑mTOR and p‑ribosomal protein S6 kinase β‑1 were significantly greater in the insulin‑treated group. These results demonstrated that although an embryo may implant into endometrium, mice endometrium receptivity in early pregnancy may be impaired by maternal hyperinsulinemia. In addition, mTOR signaling may be involved in this process. The present study provides preliminary results demonstrating that female reproduction may be compromised during hyperinsulinemia, which requires further investigation in future studies.

Benzo[a]pyrene (BaP) is a ubiquitous environmental persistent organic pollutant and a well-known endocrine disruptor. BaP exposure could alter the steroid balance in females. Endometrium decidualization and decidual angiogenesis are critical events for embryo implantation and pregnancy maintenance during early pregnancy and are modulated by steroids. However, the effect of BaP on decidualization is not clear. This study aimed to explore the effects of BaP on decidualization and decidual angiogenesis in pregnant mice. The result showed that the uteri in the BaP-treated groups were smaller and exhibited an uneven size compared with those in the control group. Artificial decidualization was detected in the uteri of the controls, but weakened decidualization response was observed in the BaP-treated groups. BaP significantly reduced the levels of estradiol, progesterone, and their cognate receptors ER and PR, respectively. The expression of several decidualization-related factors, including FOXO1, HoxA10, and BMP2, were altered after BaP treatment. BaP reduced the expression of cluster designation 34 (CD34), which indicated that the decidual angiogenesis was inhibited by BaP treatment. In addition, BaP induced the downregulation of vascular endothelial growth factor A. These data suggest that oral BaP ingestion compromised decidualization and decidual angiogenesis. Our results provide experimental data for the maternal reproductive toxicity of BaP during early pregnancy, which is very important for a comprehensive risk assessment of BaP on human reproductive health.

Benzo [a]pyrene (BaP) is a well-known endocrine disruptor. Exposure to BaP is known to impair embryo implantation. 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. However, the effect of BaP on CL remains unknown. This study investigated the deleterious effects of BaP on the structure and function of CL during early pregnancy. Pregnant rats were dosed with BaP at 0.2 mg.kg-1. d from day 1 (D1) to day 9 (D9) of gestation. We found that BaP reduced the number of CLs, disturbed the secretion of steroid and impacted the luteal vascular networks. BaP significantly decreased the angiogenesis factor (VEGFR, Ang-1 and Tie2) and increased the anti-angiogenic factor THBS1. Inhibited THBS1 function by LSKL partially rescued the angiogenesis defect caused by BaP. In vitro, BaP metabolite BPDE also interfered the expression levels of angiogenesis-related factors in HUVECs and impaired the angiogenesis, whereas supplemented with rAng-1 can alleviate the anti-angiogenic effect of BPDE. Furthermore, Notch signaling molecules, including Notch1, Dll4, Jag1 and Hey2, which are essential for the establishment and maturation of vascular networks, were affected by BaP exposure. Collectively, BaP broke the molecular regulatory balance between luteal angiogenesis and vascular maturation, impaired the construction of luteal vascular networks, and further affected luteal formation and endocrine function during early pregnancy. Our findings might provide new insight into the relationship between BaP and luteal insufficiency in early pregnancy. These data also give a new line of evidence for curtailing BaP emissions and protecting the women of childbearing age from occupational exposure.

Embryo implantation is an essential and complex process in mammalian reproduction. However, little evidence has indicated the involvement of autophagy during embryo implantation. To determine the possible role of autophagy in uterine of pregnant mice during the peri-implantation stage, we first examined the expression of autophagy-related markers ATG5 and LC3 on day 4, 5, and 6 of pregnancy (D4, D5, and D6, respectively). Compared with expression on D4, downregulation of the autophagy-related markers was observed on D5 and D6, the days after the embryo attached to the receptivity endometrium. Further examination showed that autophagy-related markers ATG5, ATG12, LC3, cathepsin B, and P62 at the implantation site were significantly decreased when comparing with the inter-implantation site. Fewer number of autophagosomes at the implantation site were also observed by transmission electron microscopy. To confirm the functional role of autophagy during embryo implantation in mice, we administered the autophagy inhibitor 3-methyladenine and chloroquine to mice. After treated with 3-methyladenine, the expression of decidual markers HOXA10 and progesterone receptor were significantly reduced. Furthermore, a reduction in implantation sites and increase in the HOXA10 and PR protein levels were observed in response to chloroquine treatment. In addition, impaired uterine decidualization and dysregulation of the PR and HOXA10 protein levels was observed after autophagy inhibited by 3-methyladenine and chloroquine in in vivo artificial decidualization mouse model. In the last, LC3 and P62 were also observed in normal human proliferative, secretory, and decidua tissues. In conclusion, endometrial autophagy may be essential for embryo implantation, and it may be associated with endometrial decidualization during early pregnancy. KEY MESSAGE: • Autophagy-related markers were significantly decreased at implantation site. • Autophagy inhibition results in abnormal decidualization. • Autophagy is essential for embryo implantation.

Dibutyl phthalate (DBP), one of the most widely used plasticizers, is a known environmental endocrine disruptor that impairs male and female fertility. In this study, oral administration of DBP was given to pregnant mice on 14.5 days post coitus (dpc) for 3 days; and additionally, DBP was added into the culture of 14.5 dpc fetal ovaries for 3 days. DBP exposure during gestation disturbed the progression of meiotic prophase I of mouse oocytes, specifically from the zygotene to pachytene stages. Meanwhile, the DBP-exposed pachytene oocytes showed increased homologous recombination sites and unrepaired DNA damage. Furthermore, DBP caused DNA damage by increasing oxidative stress, decreased the expression of multiple critical meiotic regulators, and consequently induced oocyte apoptosis. Moreover, the effect of DBP on meiosis I prophase involved estrogen receptors α and β. Collectively, these results demonstrated a set of meiotic defects in DBP-exposed fetal oocytes. As aberrations in homologous recombination can result in aneuploid gametes and embryos, this study provides new support for the deleterious effects of phthalates.

Folate deficiency has been linked to a wide range of pregnancy disorders. Most research about folate-deficiency has focused on the embryo itself, little attention has been paid to possible effects on the placenta. According to our results, the morphology of the placenta, endocrine function, and the expression of genes involved in placental differentiation were all abnormal in folate-deficient mice on days 10, 12, and 14 of pregnancy. Similar results were found in human placenta explants cultured in folate-deficient medium. Autophagy is an inducible catabolic process activated by external nutrients starvation. Here we explored further, whether autophagy was involved in the abnormal placentation caused by folate-deficiency. The aberrant number of autophagosomes measured by transmission electron microscopy and the deviant expression of autophagy-related markers showed a disordered autophagy in placentas under conditions of folate-deficiency in vivo and in vitro dual-fluorescence mRFP-eGFP-LC3 analysis indicated enhanced autophagy was detected in HTR8/SVneo cells incubated in folate-deficient medium. Importantly, the placentation impairment in mice and human placenta explants could be recovered by inhibiting placental autophagy using 3-MA. In addition, the apoptosis and invasive capability of HTR8/SVneo cells were obviously suppressed by folate deficiency but notably elevated by 3-MA. These data suggest that folate deficiency can impair placentation and autophagy is a key factor in this. However, the signal pathway by which folate deficiency causes aberrant autophagy needs to be explored further.

Background/aims Excessive apoptosis of trophoblasts, induced by sustained hypoxia, leads to abnormal placentation and is strongly linked to pregnancy complications such as preeclampsia (PE). Wild-type p53-induced phosphatase (Wip1) positively regulates cellular survival in tumor cells through the p38 and p53 pathways, but its expression pattern and effects in trophoblasts have yet to be reported. This study clarified the effect of Wip1 on the regulatory mechanism of p53-dependent apoptosis in trophoblasts, and thus increases understanding of the etiology of PE. Methods In normal and PE placentas, Wip1 mRNA and protein levels were determined by RT-qPCR and Western blotting respectively, while localization of Wip1 in placental tissues and in HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Two in vitro trophoblastic PE models were established by subjecting HTR8/SVneo cells to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 was suppressed in the aforementioned PE models by specific inhibitor or shRNA, and apoptosis was then assessed by flow cytometry, while further validation was done by measurement of cleaved-caspase 9 expression by Western blotting. The p38 inhibitor SB202190, Mdm2 inhibitor NVP-CGM097, and proteasome inhibitor MG-132 were administered in PE models, either in combination or alone, to determine the regulatory order of the component signal molecules of the feedback loop. The impact of Wip1 on p53-Mdm2 interaction was examined by coimmunoprecipitation. Lastly, the upregulation of the p38-Wip1 loop was confirmed in human placentas from pregnancies complicated by PE, using Western blotting. Results Wip1 expression was significantly elevated in human PE placentas and in vitro trophoblastic PE models; this is opposite to the pattern observed in tumor cells. Inhibition of Wip1 rescued hypoxia-induced p38 activation, cleavage of caspase 9 and apoptosis but significantly compromised p53-Mdm2 binding, while p-p53Ser15 was increased. Inhibition of Mdm2 degradation resulted in p53 destabilization and p38-Wip1 loop down-regulation, while degradation of the p53-Mdm2 complex resulted in p53 accumulation and p38-Wip1 loop hyperactivation. However, the p53-Mdm2 interaction was found to be more important in the regulation of the p38-Wip1 loop than Mdm2 stability. Conclusion Trophoblastic p53 homeostasis is maintained by the p38-Wip1 feedback regulatory loop in response to hypoxic stress, which is dysregulated in the placentas of pregnancies complicated by PE, and thereby leads to excessive apoptosis.

Objective: Oxidative stress plays a significant role in the pathogenesis of preeclampsia (PE), by inducing trophoblast cell death and consequent placental dysfunction. Quiescin sulfhydryl oxidase 1 (QSOX1) is upregulated in many types of cancer cells; it promotes disulfide bond formation as well as hydrogen peroxide (H2O2) production. The aims of present study are to investigate the expression pattern of QSOX1 in placentae of pregnancies complicated by PE and the role of QSOX1 in the regulation of trophoblastic function, thus providing in-depth understanding of the putative involvement of QSOX1 in the development of PE. Methods: Human term placenta from normal pregnancies and from pregnancies complicated by PE was collected to measure QSOX1 expression and H2O2 levels. Down-regulation of QSOX1 in HTR-8/SVneo cells was achieved by siRNA interference. An in vitro cellular PE model was generated by hypoxic incubation. Protein expression levels were assessed by Western blotting, and H2O2 levels were determined in the cell culture medium as well as in the cell lysate. Trophoblast apoptosis was evaluated by TUNEL staining. Results: QSOX1 was overexpressed in the PE placenta. Inhibition of QSOX1 expression in HTR-8/SVneo cells attenuated cell apoptosis and intracellular H2O2 levels. Hypoxia-induced QSOX1 expression in HTR-8/SVneo cells and led to apoptosis of HTR-8/SVneo cells, and knock-down of QSOX1 rescued hypoxia-induced trophoblast apoptosis. Conclusions: Hypoxia-induced upregulation of QSOX1 and a consequent elevation in intracellular H2O2 increased apoptosis in placentae of pregnancies complicated by PE.

Embryo implantation is essential for normal pregnancy, and the process of decidualization is critical for embryo implantation. However, the mechanism of decidualization during early pregnancy is still unknown. Forkhead box O3a (FOXO3a) is the most important functional transcription factor of the forkhead box family and is a highly conserved transcription factor of apoptosis-related genes. In the mouse uterus, FOXO3a was found to be expressed regularly from Days 1-7 of early pregnancy. Upon further exploration, it was found that FOXO3a was expressed at significantly higher levels at the implantation site than at the interimplantation site on Days 5-7 of pregnancy. Under artificial decidualization, FOXO3a was highly expressed in the first and second decidual zones. After decidualization, the expression of FOXO3a was significantly increased both in vivo and vitro. In primary stromal cells, apoptosis was reduced by decreased expression of FOXO3a after inducing decidualization. Moreover, when FOXO3a-small interfering RNA was transfected into the uteri of mice, the expression of decidualization- and apoptosis-related factors was impaired. Thus, FOXO3a might play an important role in decidualization during early pregnancy, and cell apoptosis might be one of pathways for FOXO3a-regulated decidualization.

The female reproductive lifespan is largely determined by the size of the primordial follicle pool, which is established early in life. We previously reported that Di (2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor and a widely-spreading plasticizer, impairs primordial folliculogenesis. In the present study, we found DEHP significantly altered the number and sex ratio of the offspring of neonatal-exposed mice. Furthermore, by a neonatal exposure model and an ovary culture model, it showed that DEHP activated autophagy in the ovary, with increased autophagy-related gene expression and recognizable autophagosomes, while inhibition of autophagy by 3-MA attenuated the adverse impact of DEHP on primordial folliculogenesis. Moreover, key components of AMPK-SKP2-CARM1 signalling were up-regulated by DEHP in the ovary, and AMPK inhibitor Compound C reduced autophagy-related gene expression and partially recovered primordial follicle assembly. Collectively, this study demonstrates that DEHP induces autophagy by activating AMPK-SKP2-CARM1 signalling in mice perinatal ovaries, which results in disrupted primordial folliculogenesis and reduced female fertility.

Background/Aims: Preeclampsia (PE) has long been assumed to be an ischemic disease of the placenta, although there is limited evidence as to how the ischemia impacts on the placenta. AMP-activated protein kinase (AMPK) is a key regulator of cellular energy metabolism and plays an important role in a variety of ischemic diseases by enhancing energy production. The present study investigated placental metabolism in PE, and the role of AMPK in regulating trophoblast function. Methods: placentas from normal and PE complicated pregnancies were subjected to GC-MS to identify fatty acids (FA) metabolic fingerprints, and total FA oxidation was assessed by malondialdehyde (MDA) measurement. The AMPK-ACC signaling pathway was assessed by q-PCR and Western Blotting. HTR8/SVneo trophoblast cultures were exposed to different oxygenation conditions to establish an in vitro PE cell model; further analysis by GC-MS for metabolite profiling was then undertaken. Trophoblasts invasion was assessed by a matrigel transwell assay in the presence/absence of AMPK expression and after manipulations of AMPK activity, and then further validated by human villi outgrowth experiments. Results: AMPK phosphorylation and MDA production were significantly elevated in placentas from pregnancies complicated by PE. Metabolism of cis double bond FA was inhibited while trans double bond FA metabolism was promoted in PE placentas. HTR8/SVneo cell culture conditions of persistent low oxygenation mimicked the hyper-activation of AMPK and enhanced the FA oxidation that was observed in PE. AMPK activation impaired trophoblast invasion, while AMPK inhibition promoted trophoblast invasion. Conclusion: PE complicated placentas are associated with AMPK hyper-activation and consequent alterations in FA oxidation, which inhibit trophoblast invasion.

Vangl1 and Vangl2 are homologous genes belonging to the group of highly conserved planar cell polarity proteins. It has been shown that Vangl1 and Vangl2 are essential for embryonic development, cell adhesion, migration and polarity. We examined the expression of Vangl1 and Vangl2 in the uterus of mice during early pregnancy. They are upregulated in the endometrium of peri-implantation and reached the peak on D5. Vangl1 mRNA is widely distributed in the luminal epithelium, glandular epithelium and stromal cells in the endometrium, while its protein only appeared in the stromal cells. The localization of Vangl2 protein overlapped with its mRNA. In addition, expression of Vangl1 in the endometrium of pseudopregnant mice was lower than that of pregnant mice, whereas the level of Vangl2 was not significantly different, suggesting that expression of Vangl1 is induced by embryo. Further study showed that implantation would be suppressed after silencing expressions of Vangl1 and Vangl2 by uterine injection with antisense oligonucleotides. These findings suggest that Vangl1 and Vangl2 may play a key role in the embryo implantation of mice.

Studies have shown that metabolic syndrome is associated with reproductive problems. Women with metabolic syndrome, characterized by hyperinsulinemia, have common ovarian dysfunction, but the mechanism remains elusive. The aim of this research is to explore the effects of high levels of insulin on ovary function during early pregnancy. Mice in the high insulin-exposed group were given a subcutaneous injection of human recombinant insulin. After insulin treatment, changes in various hormones were tested using ELISA kits which showed hormones secreted by the ovary were significantly altered in the insulin group. TUNEL staining showed less TUNEL-positive cells in the insulin group. A significant decrease in Bax and an increase in Bcl2 in the ovary were found in the insulin group by immunohistochemical studies. Western blotting showed the expressions of apoptosis related proteins in the ovaries from the insulin group were obviously altered. In addition, expression of p-Akt proteins in the ovaries from the insulin group was significantly upregulated. Moreover, the Akt inhibitor LY294002 reversed the anti-apoptotic effects of high insulin in the ovary tissues in early pregnancy mice. All of these results showed that insulin impaired ovarian function during early pregnancy and ovarian apoptosis is imbalanced under the role of insulin. The PI3K/AKT signalling pathway might participate in this process.

Ubiquitination is a regulatory mechanism that occurs after protein translation. To date, few studies have reported on ubiquitination during embryo implantation. We used real-time quantitative polymerase chain reaction, immunohistochemistry, and Western blotting analyses to analyze the expression of speckle-type pox virus and zinc finger (POZ) protein (SPOP; an adapter of E3 ligases of ubiquitination) in mouse uteri during early pregnancy and pseudopregnancy using an artificially induced decidualization model and a steroid hormone–processing model. At the same time, we established an artificially induced decidualization in vitro model. We observed that SPOP regulates endometrial stromal cell decidualization in mice and that hormones regulate the expression of SPOP. This study suggests that ubiquitination may be involved in embryonic implantation.

STUDY QUESTION Does nm23 have functional significance in decidualization in mice and humans? SUMMARY ANSWER nm23 affects decidualization via the phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathways in mouse endometrial stromal cells (ESCs; mESCs) and human ESCs. WHAT IS KNOWN ALREADY The function of nm23 in suppressing metastasis has been demonstrated in a variety of cancer types. nm23 also participates in the control of DNA replication and cell proliferation and differentiation. STUDY DESIGN, SIZE AND DURATION We first analyzed the expression profile of nm23 in mice during early pregnancy (n = 6/group), pseudopregnancy (n = 6/group) and artificial decidualization (n = 6/group) and in humans during the menstrual cycle phases and the first trimester. We then used primary cultured mESCs and a human ESC line, T-HESC, to explore the hormonal regulation of nm23 and the roles of nm23 in in vitro decidualization, and as a possible mediator of downstream PI3K-Akt-mTOR signaling pathways. PARTICIPANTS/MATERIALS, SETTINGS AND METHODS We evaluated the dynamic expression of nm23 in mice and humans using immunohistochemistry, western blot and real-time quantitative RT-PCR (RT-qPCR). Regulation of nm23 by steroid hormones was investigated in isolated primary mESCs and T-HESCs by western blot. The effect of nm23 knockdown (using siRNA) on ESC proliferation was analyzed by 5-ethynyl-2'-deoxyuridine staining (EdU) and proliferating cell nuclear antigen protein (PCNA) expression. The influence of nm23 expression on the differentiation of ESCs was determined by RT-qPCR using the mouse differentiation markers decidual/trophoblast PRL-related protein (dtprp, also named prl8a2) and prolactin family 3 subfamily c member 1 (prl3c1) and the human differentiation markers insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL). The effects of nm23 siRNA (si-nm23) and the PI3K inhibitor LY294002 on the downstream effects of nm23 on the PI3K-Akt-mTOR signaling pathway were estimated by western blot. MAIN RESULTS AND THE ROLE OF CHANCE NM23-M1 was specifically expressed in the decidual zone during early pregnancy and in artificially induced deciduoma, and NM23-H1 was strongly expressed in human first trimester decidua. The expression of nm23 was upregulated by oestradiol and progesterone (P

Mammalian target of rapamycin (MTOR) is a protein kinase that plays a central role in cell growth and proliferation. It is a part of the signaling network transmitting growth factor signaling to translational control. Previous studies have shown that MTOR is involved in embryo implantation, but its expression in the uterus and its role in implantation are unclear. Here, we have investigated the expression and role of MTOR in mouse uterus during early pregnancy. RT-FQ PCR showed that the mRNA levels of Mtor in endometria of pregnant mice were higher than those of nonpregnant mice. The mRNA levels in the pregnant mice gradually increased from D3 of pregnancy, reached maximum on D5, and then declined afterward. In situ hybridization and immunohistochemical analysis showed that the mRNA and protein of MTOR were mainly located in stromal cells. The levels of the expressed MTOR protein correlate with those of mRNA. The number of implantation sites was greatly decreased by the intrauterine injection with rapamycin in the early morning of D4 of the pregnancy. These findings suggest that MTOR may play an important role in embryo implantation in mice.

The mechanism of birth defects induced by folate deficiency was focused on mainly in fetal development. Little is known about the effect of folate deficiency on the maternal uterus, especially on decidual angiogenesis after implantation which establishes vessel networks to support embryo development. The aim of this study was to investigate the effects of folate deficiency on decidual angiogenesis. Serum folate levels were measured by electrochemiluminescence. The status of decidual angiogenesis was examined by cluster designation 34 (CD34) immunohistochemistry and the expression of angiogenic factors, including vascular endothelial growth factor A (VEGFA), placental growth factor (PLGF), and VEGF receptor 2 (VEGFR2) were also tested. Serum levels of homocysteine (Hcy), follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), progesterone (P4), and estradiol (E2) were detected by Enzyme-linked immunosorbent assay. The folate-deficient mice had a lower folate level and a higher Hcy level. Folate deficiency restrained decidual angiogenesis with significant abnormalities in vascular density and the enlargement and elongation of the vascular sinus. It also showed a reduction in the expressions of VEGFA, VEGFR2, and PLGF. In addition, the serum levels of P4, E2, LH, and PRL were reduced in folate-deficient mice, and the expression of progesterone receptor (PR) and estrogen receptor α (ERα) were abnormal. These results indicated that folate deficiency could impaire decidual angiogenesis and it may be related to the vasculotoxic properties of Hcy and the imbalance of the reproductive hormone.

Existing evidence suggests that adverse pregnancy outcomes are closely related with dietary factors. Previous studies in mice have focused on the harm of folate deficiency (FD) on development of embryo, while the effect of low maternal folate levels on maternal intrauterine environment during early pregnancy remains unclear. Since our previous study found that FD treatment of mice causes no apparent defects in embryo implantation but is accompanied by female subfertility, we next chose to investigate a potential role of FD on molecular events after implantation. We observed that the decidual bulges began to be stunted on pregnancy day 6. The results of functional experiments in vivo and in vitro showed that FD inhibited the process of endometrial decidualization. It has been confirmed that DNA methylation participates in decidualization, and folate as a methyl donor could change the methylation patterns of genes. Thus, we hypothesized that FD impairs maternal endometrial decidualization by altering the methylation profiles of related genes. Reduced representation bisulphite sequencing was carried out to detect the methylation profiles of endometrium on pregnancy day 6-8, which is equivalent to the decidualization period in mice. The results confirmed that FD changes the methylation patterns of genome, and GO analysis of the differentially methylated regions revealed that the associated genes mainly participate in biological adhesion, biological regulation, cell proliferation, development, metabolism and signalling. In addition, we found some candidates for regulators of decidual transformation, such as Nr1h3 and Nr5a1. The data indicate that FD inhibits decidualization, possibly by altering methylation patterns of the genome in mice.

STUDY QUESTION Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans. SUMMARY ANSWER The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered β1,6-N-acetylglucosamine (β1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin β1. WHAT IS KNOWN ALREADY GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported. STUDY DESIGN, SIZE, DURATION Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM. PARTICIPANTS, MATERIALS, SETTING, METHODS Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of β1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5β1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the β1,6-GlcNAc and bis-GlcNAc structures in integrin α5β1 during the first trimester in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the normal group expression and activity of GnT-V and the concentration of its product, β1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and β1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. β1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased β1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin β1 (P < 0.05) were observed in the ESM group. WIDER IMPLICATIONS OF THE FINDINGS Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM. LIMITATIONS, REASONS FOR CAUTION The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests.

Benzo(a)pyrene (BaP) is a ubiquitous environmental pollutant. Studies have demonstrated it to be an endocrine-disrupting chemical that can cause adverse effects on the female reproductive system. However, the effect of BaP on early pregnancy has not been reported. We investigated the effect of BaP on endometrial receptivity and embryo implantation. Pregnant mice were dosed with BaP at 0.2, 2 and 20 mg/kg/day from day 1 (D1) to day 5 (D5) of gestation. Exposure to BaP impaired the morphology of the endometrium and decreased the number of implantation sites (p0.2=0.006, p2=0.167, p20=0.003). Levels of estrodiol (p0.001, for three treatment group compare with control group) and progesterone-4 in plasma were elevated in BaP-treatment groups (p0.20.001, p20.001, p20=0.032). Expression of estrogen receptor-α was up-regulated (p0.2=0.002, p2=0.131, p20=0.024) whereas expression of the progesterone receptor was down-regulated (p0.20.001, p2=0.064, p20=0.021). Levels of receptivity-related genes HoxA10 (p0.20.001, p2=0.135, p200.001) and E-cadherin (p0.2=0.002, p2=0.624, p20=0.137) were changed by BaP. These results revealed that BaP can disrupt the balance of estrogen and progesterone, influence expression of their receptors and downstream related genes, lead to changes in endometrium receptivity, and reduce of the number of implantation sites.

Background Folate, one of the B vitamins, provides the one-carbon units required for methylation. Folate deficiency has been associated with many pathologies. However, much less is known about the effect of it on human reproduction, especially on implantation. The establishment of uterine receptivity is crucial for successful embryo implantation. Gene expression can be influenced by both heredity and epigenetics such as DNA methylation. However, it is not known whether the methylation and expression of genes related to uterine receptivity can be affected by folate levels. To explore whether folate deficiency affected the epigenetic regulation of genes related to uterine receptivity, and their influence on implantation, we investigated the methylation and expression of cadherin 1 (Cdh1), progesterone receptor (Pgr) and estrogen receptor 1 (Esr1) genes during implantation and the implantation efficiency using a folate-deficient pregnant mouse model. Methods Serum folate levels of pregnant mice were measured using the electro-chemiluminescence immunoassay. The methylation status of Cdh1, Pgr and Esr1 promoter regions was determined by methylation-specific PCR and bisulfite sequencing. The expression of Cdh1, Pgr and Esr1 in the implantation-site endometrium was examined by real-time PCR, western blot and immunohistochemistry. The number and the morphology of pinopodes, important morphological markers of endometrial receptivity, were examined using scanning electron microscopy. The number of implantation sites demarcated by distinct blue bands was recorded. Results Serum folate levels of the folate-deficient group were lower (5.42 ± 1.35 ng/ml, n= 42) than those of the normal group (24.13 ± 4 .26 ng/ml, n= 37; P = 0.003). Here we show that the methylation status and mRNA levels of Esr1 were decreased (P= 0.021, P= 0.045, respectively), while the Cdh1 and Pgr expression levels were slightly but not significantly elevated and the methylation status did not vary in the folate-deficient mice compared with the wild type. Neither the number nor morphology of pinopodes was affected by folate deficiency. Furthermore, folate deficiency did not affect the number of implantation sites in mice. Conclusions This study demonstrates for the first time that, unlike the effects on Esr1, folate deficiency in mice does not influence the methylation and expression of Pgr and Cdh1, two genes shown to be essential for uterine receptivity and embryo implantation. Embryo implantation in mice appears to be unaffected by a deficiency in folate, suggesting that abnormalities in a pregnancy caused by folate deficiency start to develop after implantation.

Di-(2-ethylhexyl)-phthalate (DEHP) is a ubiquitous environmental pollutant and endocrine disruptor (ED) that causes serious adverse effects on animal and human health. The harmful effects of DEHP on human reproduction are increasingly recognized, especially in women. However, it is not known how endometrial receptivity and embryo implantation, which play important roles in the establishment of pregnancy, are affected by DEHP. This study was aimed towards investigating the effects of DEHP on endometrial receptivity and embryo implantation in pregnant mice. The pregnant mice received DEHP at 0, 250, 500 and 1000 mg/kg/day from day 1 (D1) of gestation until sacrifice. Administration of DEHP led to compromised endometrial receptivity and decreased number of implantation sites. The mRNA and protein expression levels of ERα, PR and E-cadherin, but not those of HoxA10 and MMP-2, were up-regulated by DEHP in the mouse endometrium. The results further suggested that DEHP disrupts the MAPK and NF-κB signaling pathways. This was maybe one of paths which influenced the E-cadherin expression. In conclusion, DEHP reduced endometrial receptivity and impaired embryo implantation by influencing the expression of hormone receptors and E-cadherin. Therefore, determining the full extent of the hazards of DEHP to human reproduction will be vital to developing and implementing effective protective measures.

The expression of IK cytokine was investigated in the mouse endometrium during early pregnancy (D1-D7 of pregnancy) and pseudopregnancy using real-time PCR, western blotting and immunohistochemical analysis, and the effects of IK cytokine on embryo implantation were observed by injection with antisense IK cytokine oligodeoxynucleotides in the uterine horn. Our data showed that the expression of IK cytokine mRNA increased gradually from D1 to D4 of pregnancy and reached a peak level at D4 of pregnancy (P

Calreticulin (CRT), a Ca 2+ -binding storage protein and chaperone in the endoplasmic reticulum, modulates cell adhesiveness and integrin-dependent Ca 2+ signaling. However, the role of CRT during implantation remains poorly understood. In the present study, we characterized the expression of CRT mRNA and the protein in mouse endometria from pregnancy D1 to D7. Real-Time PCR and in situ hybridization results showed that the levels of CRT mRNA in the endometria of pregnant mice were significantly higher than those of non-pregnant mice (P

OBJECTIVE: Embryo implantation is directly affected by genes related to uterine receptivity. Studies have demonstrated the important roles of miRNAs in the regulation of gene expression. Our early miRNA chip analyses revealed that the mmu-miR-141 expression in endometrial tissue is lower after embryo implantation than before it. However, the possible roles of miR-141 in embryo implantation have not yet been elucidated. Here, mmu-miR-141 was designed to detect the expression and role of miR-141 in the endometria of mice in early pregnancy following embryo implantation. METHODS: Real-time PCR and in-situ hybridization were used to study mmu-miR-141 expression in mouse uterus. Cell proliferation was detected by tetrazolium dye (MTT) assay and flow cytometry. Real-time PCR and Western blot analysis were used to confirm the mRNA and protein levels of phosphatase and tensin homolog (PTEN) to determine whether it was the target gene of mmu-miR-141. Enhanced green fluorescent protein (EGFP) fluorescence reporter vector analysis was also performed. A functional study was performed by injecting mice uteri with mmu-miR-141 inhibitor or mimic vectors. RESULTS: mmu-miR-141 expression was lower on day 6 (D6) than day 4 (D4) and could be increased by progesterone. Reduced mmu-miR-141 could decrease the proliferation activity of stromal cells and promote apoptosis. Upregulation of mmu-miR-141 inhibited PTEN protein expression but downregulation of mmu-miR-141 increased it, while the mRNA level remained unchanged. EGFP fluorescence reporter vector analysis showed that miR-141 targets the 3'-untranslated region of the PTEN mRNA. In addition, when the physiological mmu-miR-141 level was altered on D2 by injecting with inhibitor or mimic, the embryo implantation sites were significantly decreased on D7. CONCLUSIONS: This study demonstrated that mmu-miR-141 might influence cell proliferation and apoptosis in the endometrium by negatively regulating PTEN expression, and could also influence the number of embryo implantation sites. mmu-miR-141 plays an essential role in embryo implantation.