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Purpose: This study aimed to compare the effect of gonadotropin-releasing hormone agonist (GnRHa) trigger alone versus dual trigger comprising GnRHa and low-dose human chorionic gonadotropin (hCG) on reproductive outcomes in patients with polycystic ovary syndrome (PCOS) who received the freeze-all strategy. Methods: A total of 615 cycles were included in this retrospective cohort study. Propensity score matching (PSM) was performed to control potential confounding factors between GnRHa-trigger group (0.2 mg GnRHa) and dual-trigger group (0.2 mg GnRHa plus 1000/2000 IU hCG) in a 1:1 ratio. Multivariate logistic regression was applied to estimate the association between trigger methods and reproductive outcomes. Results: After PSM, patients with dual trigger (n = 176) had more oocytes retrieved, mature oocytes, and 2PN embryos compared to that with GnRHa trigger alone. However, the oocytes maturation rate, normal fertilization rate, and frozen embryos between the two groups were not statistically different. The incidence of ovarian hyperstimulation syndrome (OHSS) (14.8% vs. 2.8%, P < 0.001) and moderate/severe OHSS (11.4% vs. 1.7%, P < 0.001) were significantly higher in dual-trigger group than in GnRHa-alone group. Logistic regression analysis showed the adjusted odds ratio of dual trigger was 5.971 (95% confidence interval 2.201–16.198, P < 0.001) for OHSS. The pregnancy and single neonatal outcomes were comparable between the two groups (P > 0.05). Conclusion: For PCOS women with freeze-all strategy, GnRHa trigger alone decreased the risk of OHSS without damaging oocyte maturation and achieved satisfactory pregnancy outcomes. [ABSTRACT FROM AUTHOR]

Purpose: As a member of the C19MC family, miR-526b-5p is mainly expressed in the placental tissue and is a well-known tumor suppressor microRNA. However, its effect on the function of trophoblasts and its role in the development of recurrent spontaneous abortion (RSA) remains unclear. Methods: Transcriptome sequencing, quantitative real-time polymerase chain reaction (RT-qPCR), Western blot, 5-ethynyl-2′-deoxyuridine (Edu) proliferation analysis, cell counting kit-8 (CCK8) assay, Transwell assays, and wound healing were used to detect the proliferation, migration, and invasion capacity of trophoblasts. Target genes of miR-526b-5p were obtained by the dual luciferase reporter system. The promoter-reporter system and ChIP-qPCR were used to prove that c-Myc positively regulated the expression of Foxp1 Results: The miR-526b-5p levels were significantly higher in patients with RSA than in controls. High expression of miR-526b-5p inhibited the proliferation, migration, and invasion of trophoblast cell line. By contrast, low expression of miR-526b-5p promoted the proliferation and migration of trophoblast cell line. Target genes of miR-526b-5p were c-Myc and Foxp1. c-Myc positively regulated the expression of Foxp1 by binding to the Foxp1 promoter location -146/-135. Finally, miR-526b-5p impeded the proliferation, migration, and invasion of trophoblasts by negatively regulating c-Myc by rescue experiments. Conclusion: Thus, miR-526b-5p affected the proliferation, migration, and invasion of trophoblasts by targeting c-Myc and Foxp1. Low expression of c-Myc further deactivated the positive transcriptional regulation of c-Myc on Foxp1, which may be the mechanism of RSA. This study provides potential therapeutic targets and clues for the diagnosis and treatment of RSA. [ABSTRACT FROM AUTHOR]

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. Graphical highlights: 1. Dnmt3bd/d female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. 2. Losing Dnmt3b did not alter genomic methylation patterns of the decidual endometrium during early pregnancy. 3. Pgk1 was one of the most variable genes in Dnmt3bd/d decidual endometrium and the compromised decidualization upon the downregulation of Dnmt3b was related to the overexpression of Pgk1. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Women with hyperinsulinism and insulin resistance have reduced fertility, but the underlying mechanism is still poorly understood. Aberrant endometrial decidualization in early pregnancy was linked to pregnancy complications. In this study, we aimed to test whether elevated insulin levels compromise decidualization in early-stage pregnancy. C57BL/6J mice in high insulin-exposed group were given a subcutaneous injection of recombinant insulin at a concentration of 0.05 IU daily. During decidualization in early pregnancy, serum levels of insulin, E2, P4, LH, FSH and blood glucose were significantly altered in mice treated with high insulin levels. The number of embryo implantation sites and endometrial decidual markers BMP2, ER, PR was significantly decreased by high insulin levels in vivo. Artificial decidual induction in primary mouse endometrial stromal cells and immortal human endometrial stromal cells line were all compromised after treated with 100 nmol/L insulin levels. All these results on flow cytometry, transmission electron microscopy and western blotting of Bax, Bcl2, cleaved Caspase3, cleaved PARP proteins level showed that decidual cells apoptosis was significantly decreased. Mitochondrial transmembrane potential also significantly increased by the influence of high insulin levels. PI3K and p-Akt were much higher after insulin exposure and the compromised decidualization by high insulin treatment was rescued by PI3K/Akt inhibitor LY294002 both in vitro and in vivo. In conclusion, we demonstrated that elevated insulin levels could compromise mice decidualization in early-stage pregnancy and PI3K/p-Akt-regulated apoptosis was essential for this role. It provides a clue for future investigation on compromised reproduction in women with hyperinsulinemia. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a hypertensive disease associated with increased endothelial cell dysfunction caused by systemic oxidative stress. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that are upregulated in several types of cancer. However, its role in PE remains unclear. In this study, we found that ACTN4 was localized in placenta vascular endothelial cells (ECs), and its expression was downregulated in primary human umbilical vein endothelial cells (HUVECs) from severe preeclamptic patients compared to that in HUVECs from normotensive pregnant women. ACTN4 expression was also decreased in normotensive HUVECs treated with H2O2. Downregulation of ACTN4 by siRNA or H2O2 treatment promoted normotensive HUVEC apoptosis and increased p38-MAPK phosphorylation along with elevated levels of p53 phosphorylation, caspase cascade proteins, and bax and repressed expression of bcl-2. Conversely, upregulation of ACTN4 in PE HUVECs significantly inhibited apoptosis and decreased p38-MAPK phosphorylation compared to that of the PE HUVEC controls. In addition, overexpression of ACTN4 in normotensive HUVECs attenuated H2O2 treatment-induced apoptosis with decreased p53 phosphorylation, caspase cascade, and bax expression levels and increased expression of bcl-2 compared to that of only H2O2 treatment. Moreover, the suppression of ACTN4 induced apoptosis, which could be blocked by the p38-MAPK inhibitor SB202190. Collectively, these results demonstrate that dysregulated ACTN4 expression may be associated with PE due to its effects on endothelial cell apoptosis via the p38-MAPK/p53 apoptosis pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Previous studies have shown that microRNAs are involved in the process of implantation. They play an important role in cell growth and proliferation. DROSHA is the microRNA-processing enzyme and is required for the maturation of microRNAs. However, its expression and function during early pregnancy in mice still remain unclear. In the present study, we analyzed the expression pattern of DROSHA in the mouse uterus during early pregnancy, pseudopregnancy, artificially induced decidualization, and in the ovariectomized mouse uterus using real-time quantitative polymerase chain reaction, Western blotting analyses, and immunohistochemistry. We found that DROSHA was spatiotemporally expressed in decidualizing stromal cells during early pregnancy and in pseudopregnant mice in which decidualization was artificially induced. In the ovariectomized mouse uterus, the expression of DROSHA was upregulated after progesterone treatment. In a stromal cell culture model, the expression of DROSHA gradually increased with the progression of stromal decidualization. Taken together, our findings suggest that DROSHA is involved in stromal decidualization and may play an important role in embryo implantation in mice. [ABSTRACT FROM AUTHOR]

Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy. [ABSTRACT FROM AUTHOR]

Embryo implantation is a complex process that involves synchronized crosstalk between a receptive endometrium and a functional blastocyst. It can take place only during the window of implantation, a period when a series of changes in gene expression occur in the endometrium to accept the embryo. As modulators of gene expression, microRNAs (miRNAs) have been identified as regulators of embryo implantation. To better understand how miRNAs regulate implantation and the related molecular mechanisms, we compared the expression profiles of miRNAs and messenger RNAs between implantation sites (IMs) and inter-IMs in the endometrium of pregnant mice on day 5 by microarrays. The results showed that compared with inter-IMs, 30 miRNAs were upregulated and 42 miRNAs (>2-fold) were downregulated at the IMs. By combining the results of the microarray experiments, we found that 20 upregulated pathways and 14 downregulated pathways might be subject to miRNA regulation at IMs. We also found that some miRNAs and their targets may play a key role in implantation. [ABSTRACT FROM AUTHOR]

Curcumin (CUR), a non-toxic polyphenol from Curcuma longa, has been investigated as a potential therapy with anti-inflammatory and anti-oxidative effects for Alzheimer's disease (AD), which depicts features of chronic inflammatory environment resulting in cellular death. However, it remains largely unknown whether the anti-inflammatory effect of CUR in AD is associated with its property of CpG demethylation, which is another function of CUR with the most research interest during recent years. Neprilysin (NEP, EP24.11), a zinc-dependent metallopeptidase expressed relatively low in the brain, is emerging as a potent inhibitor of AKT/Protein Kinase B. In addition, hypermethylated promoter of NEP has been reported to be associated with decreases in NEP expression. In the present study, using bisulfite-sequencing PCR (BSP) assay, we showed that the CpG sites in NEP gene were hypermethylated both in wild-type mouse neuroblastoma N2a cells (N2a/wt) and N2a cells stably expressing human Swedish mutant amyloid precursor protein (APP) (N2a/APPswe) associated with familial early onset AD. CUR treatment induced restoration of NEP gene via CpG demethylation. This CUR-mediated upregulation of NEP expression was also concomitant with the inhibition of AKT, subsequent suppression of nuclear transcription factor-κB (NF-κB) and its downstream pro-inflammatory targets including COX-2, iNOS in N2a/APPswe cells. This study represents the first evidence on a link between CpG demethylation effect on NEP and anti-inflammation ability of CUR that may provide a novel mechanistic insight into the anti-inflammatory actions of CUR as well as new basis for using CUR as a therapeutic intervention for AD. [ABSTRACT FROM AUTHOR]

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating literatures have indicated that T cells are involved in this process. The first signal mediated by T-cell receptor/CD3 complex and the second signal delivered by costimulatory molecules are essential for the differentiation of T cell into an effector cell. Expression and function of CD28, an important costimulatory molecule, during early pregnancy in mice is still unclear. In the present study, we investigated the expression pattern of CD28 in mouse uterus during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, Western blotting, in situ hybridization, and immunohistochemistry (IHC). We found that injection of the uterine horn with CD28 antisense oligodeoxynucleotides leads to a decreased number of implantation sites. The expression pattern of CD3 protein examined by IHC is similar to that of CD28. These findings suggest that CD28 participates in the process of embryo implantation in mice, which might play its role through delivering the second costimulatory signal. [ABSTRACT FROM AUTHOR]

Embryo implantation is a complicated process involving a series of endometrial changes that depend on differential gene expression. MicroRNAs (miRNAs) are important for regulation of gene expression. Previous studies have shown that miRNAs may participate in the regulation of gene expression during embryo implantation. To explore the role of endometrial miRNAs in early murine pregnancy, we used microarrays to investigate whether miRNAs were differentially expressed in the mouse endometrium on pregnancy day 4 (D4) and day 6 (D6). The results demonstrated that 17 miRNAs were upregulated and 18 were downregulated (>2-fold) in D6 endometria compared to D4. We identified that mmu-miR-193 exhibited the highest upregulation on D6, and the upregulation of mmu-miR-193 before embryo implantation could reduce the embryo implantation rate. Further, we demonstrated that mmu-miR-193 influenced embryo implantation by regulating growth factor receptor-bound protein 7 expression. In summary, our study suggests that mmu-miR-193 plays an important role in embryo implantation. [ABSTRACT FROM AUTHOR]

Krüppel-like factor 8 (KLF8) is a pivotal transcription factor expressed in the human placenta that can regulate cell invasion. The objective of this study was to assess whether a hypoxia–reoxygenation (H/R) environment affects placental KLF8 expression levels and subcellular localization and to evaluate the relationship between KLF8 levels and trophoblast invasion activity. Human first trimester villous tissues from normal pregnancies and third trimester placentas from pregnancies with or without preeclampsia (PE) were used for the detection of KLF8 expression and correlating its levels with metalloproteinase 9 (MMP-9) expression. In addition, HTR8/SVneo cells were used to mimic the effects of an H/R environment on placentas to study KLF8 expression and trophoblast invasion. The KLF8 levels, MMP-9 levels, and trophoblast invasion were similarly altered; the levels peaked at 8 to 10 weeks of gestation and declined thereafter along with oxygen tension increased from hypoxia to normoxia during early pregnancy, decreased in third trimester placentas from PE pregnancies featured by repeated H/R and HTR8/SVneo cells exposed to H/R compared with the control. Moreover, a visible reduction in KLF8 immunoreactivity was present in the nuclei of cytotrophoblast cells in human villous tissues at 11 weeks, and partial cytoplasmic accumulation of KLF8 was observed in HTR8/SVneo cells treated with H/R. In conclusion, these findings strongly suggest that H/R reduces the expression and nuclear localization of KLF8 to inhibit the trophoblast invasion by downregulating MMP-9 levels. The KLF8 may play a vital role in the pathogenesis of PE as a novel oxygen tension sensor. [ABSTRACT FROM PUBLISHER]

During the reproductive cycle, a number of genes controlling endometrial changes are regulated by DNA methylation, a common epigenetic modification. Because dietary folate affects DNA methylation, we determined whether a folate-deficient diet (FDD) alters DNA methylation in endometria of pseudopregnant mice, focusing on the homeobox A10 (Hoxa10) promoter. Mice were given an FDD or control diet for 40 to 45 days and examined on day 5 of pseudopregnancy. Compared to control mice, FDD mice had lower folate levels in liver and serum (P = .004). However, the FDD did not significantly affect DNA methylation within the cytosine–guanine dinucleotide (CpG)-rich Hoxa10 promoter, even when specific CpG sites were examined (P > .05). In endometrial tissue sections, the localization of anti-Hoxa10 staining was unchanged in FDD mice. Therefore, folate deficiency did not significantly affect promoter methylation or expression of Hoxa10. [ABSTRACT FROM AUTHOR]

A supramolecular fluorescent sensor of F− based on intermolecular energy transfer is described. The maximum absorption wavelength of a pyrrolic compound 1 is 472 nm, which is coincident with the emission wavelength of a dipyridylamine-anthracene compound 2. In the CH2Cl2 solution of 1 and 2, the fluorescence of 2 was quenched because of the presence of intermolecular energy transfer from 2 to 1. When F− was added to this solution, the absorption maximum wavelength of 1 shifted from 472 to 594 nm due to a deprotonation process. Simultaneously, the fluorescence of 2 was recovered because of the interruption of the intermolecular energy transfer. Based on these observations, the combination of 1 and 2 can be regarded as a novel supramolecular fluorescence enhancing F− probe. [ABSTRACT FROM AUTHOR]