Effect of 2-deoxyglucose-mediated inhibition of glycolysis on migration and invasion of HTR-8/SVneo trophoblast cells.

The proper invasion of trophoblasts is crucial for embryo implantation and placental development, which is helpful to establish a correct maternal-fetal relationship. Trophoblasts can produce a large amount of lactate through aerobic glycolysis during early pregnancy. Lactate creates a low pH microenvironment around the embryo to help uterine tissue decompose and promote the invasion of trophoblasts. The purpose of this study is to reveal the the potential mechanism of aerobic glycolysis regulating the invasiveness of trophoblasts by investigating the effect of 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, on the biological function of HTR-8/SVneo trophoblast cells, the expressions of epithelial mesenchymal transformation (EMT) markers and invasion-related factors. 2-DG could inhibit the aerobic glycolysis of trophoblasts and decrease the activity of trophoblasts in a dose-dependent manner. Moreover, 2-DG inhibited the EMT of HTR-8/SVneo cells, down-regulated the expression of invasion-related factors matrix metalloproteinase 2/9 (MMP2/9) and up-regulated the expression of tissue inhibitor of matrix metalloproteinases 1/2 (TIMP1/2), thus inhibiting cell migration and invasion. This paper provides a foundation in the significance of aerobic glycolysis of trophoblasts in the process of invasion, and also provides ideas and insights for the promotion of embryo implantation.

Neferine mediated TGF-ß/ERK signaling to inhibit fibrosis in endometriosis.

OBJECTIVE: To investigate the mechanism of Neferine in treating endometriosis fibrosis by TGF-ß/ERK signaling pathway through a combination of network pharmacological analysis of Lotus embryos, in vivo animal experiments, and in vitro cell experiments. METHODS: The active ingredients of the drug lotus embryos, the drug targets and the targets of endometriosis were determined from the TCMSP database, the Swiss Target Prediction database and GeneCard and Online Mendelian Inheritance in Man. The String database and Cytoscape 3.6.3 software were used to construct the network of common target protein interactions between drug and disease, as well as the target network. GO and KEGG enrichment analysis of the common targets was performed. We designed endometriosis mouse models with Neferine to investigate the therapeutic effect of Neferine on the fibrosis model of endometriosis and its mechanism of action. Different methods were used to evaluate the treated endometriotic lesion tissue and the untreated ectopic lesion tissue. The 12Z cells (human endometriosis immortalized cells) were cultured in vitro and treated with Neferine to detect cell viability and the effects of invasion and metastasis. RESULTS: The results of GO function and KEGG enrichment analysis showed that the role pathways of lotus germ were TGF-ß signaling pathway, ERK1/2 signaling pathway, IL-17 signaling pathway, TNF signaling pathway, AGE-RAGE signaling pathway, and PI3K-Akt signaling pathway. Neferine which is one of the effective active ingredients of lotus germ, significantly inhibited the expression of fibronectin, collagen I, connective tissue growth factor, and smooth muscle actin by activating the TGF-ß/ERK pathway in vivo, which is required for the fibrosis process of endometriosis. Neferine also significantly inhibited the proliferation, invasion and metastasis ability of 12Z cells. CONCLUSION: Neferine inhibits the progression of endometriosis both in vitro and in vivo. Its mechanism of action may involve the regulation of the TGF-ß/ERK signaling pathway, leading to the inhibition of fibrosis in endometriosis.

Shoutai Wan regulates glycolysis imbalance at the maternal-fetal interface in threatened abortion mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Threatened abortion is a common disease among women of childbearing age. Its high incidence rate and unclear etiology, seriously threaten women's physical and mental health. Shoutai Wan (STW) is a traditional Chinese medicine decoction for treating abortion. It has a long history of treating threatened abortion by tonifying the kidney and calming the fetus. However, the mechanism of STW remains unclear. AIM OF STUDY: To study the mechanism and potential benefit of STW in pregnant mice with hydrocortisone and mifepristone-induced threatened abortion. MATERIALS AND METHODS: The STW compounds were identified using gas chromatography-mass spectrometry analysis. STW-H, STW-M, or STW-L was separately given 3 mg/ml, 1.5 mg/ml and 0.75 mg/ml STW in the morning, and 2 mg/ml hydrocortisone in the afternoon from gestation day (D) 1-9 and once with 0.4 mg/kg mifepristone on D10. Didroxyprogesterone (0.1 mg/ml) and equal dose pure water were used to replace STW in didroxyprogesterone (DYD) group and model group respectively. The control group used pure water to replace STW, hydrocortisone, and mifepristone. We performed morphological and histological analyses of the maternal-fetal interface on day 10. RESULTS: The embryo loss rate in the STW-H and DYD groups was lower than that in the model group. Hematoxylin and eosin (HE) staining suggested that the morphology of maternal-fetal interface was improved in the STW-H and DYD groups. Immunohistochemical (IHC), Quantitative Reverse Transcription Polymerase Chain Reactionstaining (qRT-PCR), and Western blot (WB) results indicated that HIF-1α expression in the maternal-fetal interface of the STW-H and DYD groups was higher than that in model group. The activities of HK, PKM, LDH and the concentration of lactic acid in the STW-H and DYD groups were higher than those in model group. Furthermore, the protein and mRNA levels of HK2, PKM2, LDHA, MCT4, and GPR81 were higher in the STW-H and DYD groups than those in the model group. CONCLUSIONS: STW can reduce the pregnancy loss rate by regulating the glycolysis balance at the maternal-fetal interface of kidney deficiency threatened abortion model mice.

Label-free based quantitative proteomics analysis to explore the molecular mechanism of gynecological cold coagulation and blood stasis syndrome.

Cold coagulation and blood stasis (CCBS) syndrome is one of the common traditional Chinese medicine (TCM) syndromes of gynecological diseases. However, the molecular mechanism of CCBS syndrome is still unclear. Thus, there is a need to reveal the occurrence and regulation mechanism of CCBS syndrome, in order to provide a theoretical basis for the treatment of CCBS syndrome in gynecological diseases. The plasma proteins in primary dysmenorrhea (PD) patients with CCBS syndrome, endometriosis (EMS) patients with CCBS syndrome, and healthy women were screened using Label-free quantitative proteomics. Based on the TCM theory of "same TCM syndrome in different diseases," the differentially expressed proteins (DEPs) identified in each group were subjected to intersection mapping to obtain common DEPs in CCBS syndrome. The DEPs of gynecological CCBS syndrome in the intersection part were again cross-mapped with the DEPs of gynecological CCBS syndrome obtained by the research group according to the TCM theory of "different TCM syndromes in same disease" theory in the early stage, so as to obtain the DEPs of gynecological CCBS syndrome that were shared by the two parts. The common DEPs were subjected to bioinformatics analysis, and were verified by enzyme-linked immunosorbent assay (ELISA). A total of 67 common DEPs were identified in CCBS syndrome, of which 33 DEPs were upregulated and 34 DEPs were downregulated. The functional classification of DEPs involved in metabolic process, energy production and conversion, immune system process, antioxidant activity, response to stimulus, and biological adhesion. The subcellular location mainly located in the cytoplasm, nucleus, and extracellular. Gene ontology (GO) enrichment analysis showed that the upregulated DEPs mainly concentrated in lipid transport, cell migration, and inflammatory reaction, and the downregulated DEPs mostly related to cell junction, metabolism, and energy response. Protein domain enrichment analysis and clustering analysis revealed that the DEPs mainly related to cell proliferation and differentiation, cell morphology, metabolism, and immunity. The Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis clustering analysis showed that the upregulated DEPs were involved in inflammation and oxidative damage, while the downregulated DEPs were involved in inflammation, cell adhesion, cell apoptosis, and metabolism. The results of ELISA showed significantly increased levels of Cell surface glycoprotein MUC18 (MCAM) and Apolipoprotein C1 (APOC1), and significantly decreased levels of Vasodilator-stimulated phosphoprotein (VASP), Fatty acid-binding protein 5 (FABP5), and Vinculin (VCL) in patients with CCBS syndrome compared with healthy women. We speculated that cold evil may affect the immune process, inflammatory response, metabolic process, energy production and conversion, oxidative damage, endothelial cell dysfunction, and other differential proteins expression to cause CCBS syndrome in gynecological diseases.

Repeated controlled ovarian stimulation-induced ovarian and uterine damage in mice through the PI3K/AKT signaling pathway.

The effects of repeated controlled ovarian stimulation (COS) on the female reproductive system are still controversial. This study investigated the effects of repeated COS on the ovaries and uterus of mice and its possible mechanism. Female ICR (Institute of Cancer Research) mice were subjected to the COS using pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) for 1, 3, 5, and 7 cycles. Serum hormone levels, reactive oxidative stress (ROS), 8-hydroxy-2'-deoxyguanosine (8-OHdG), total antioxidant capacity (T-AOC), and superoxide dismutase (SOD) in the mouse ovary and uterus were analyzed by ELISA. The morphology of the ovary and endometrium, ovarian apoptosis, and expressions of the vascular endothelial growth factor (VEGF), leukemia inhibitory factor (LIF), PI3K, AKT, Bax, and Bcl-2 in the ovarian and uterine tissues were tested by hematoxylin-eosin (HE) staining, immunohistochemistry, and western blot. The results showed that repeated COS significantly decreased the hormone level (estradiol, progesterone and anti-Müllerian hormone), high-quality of the MII oocyte ratio, oocyte and embryo number, antioxidant capacity (T-AOC, SOD activity), and the protein level of Bcl-2, LIF, and VEGF, but increased the oxidative damage (ROS, 8-OHdG content), embryo fragment ratio, and expression of pro-apoptotic protein Bax. In addition, the expressions of p-PI3K and p-AKT also decreased with the increase of COS cycle. In conclusion, repeated COS causes ovarian and uterus damage possibly through the PI3K/AKT signaling pathway, and this finding may provide some experimental basis for guiding clinical treatment.

Shoutai Wan Improves Embryo Survival by Regulating Aerobic Glycolysis of Trophoblast Cells in a Mouse Model of Recurrent Spontaneous Abortion.

Background: During embryo implantation, the blastocyst exhibits a high capacity for aerobic glycolysis, which results in a unique microenvironment of high lactate/low pH at the maternal-fetal interface. Shoutai Wan (STW) is an effective Chinese herbal formula widely used in the clinical treatment of recurrent spontaneous abortion (RSA). However, the specific molecular mechanism by which STW prevents abortion is yet to be elucidated. Methods: Female CBA/J mice were allocated into six groups randomly and then mated with BALB/c mice as the control group, DBA/2 mice as the RSA model, CBA/J×DBA/2 mice treated with dydrogesterone as the DQYT group, or CBA/J×DBA/2 mice treated with low, medium, and high-dose STW as the STW-L, STW-M, and STW-H groups, respectively. Drug administration started 14 days before mating and ended on the 14th day of pregnancy. The embryo loss rate of each group was calculated on day 14 of gestation, and the pregnancy outcomes of the mice in each group were observed. The mouse serum was collected to determine the levels of progesterone (P) and chorionic gonadotropin (CG). The activities of HK2, PKM2, and LDHA, the key glycolytic enzymes in each group, were detected. The expressions of lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, and GPR81 as well as the morphology of trophoblast cells were examined. Results: The embryo loss rate and adverse pregnancy outcomes were significantly increased (P < 0.05) in the RSA model group. After dydrogesterone or different doses of STW treatment, the embryo loss rate and adverse pregnancy outcomes were rescued to varying degrees (P < 0.05). Interestingly, there was no significant difference among the groups in terms of serum P and CG (P < 0.05). Moreover, the activities of key glycolytic enzymes, lactate, ATP, HK2, PKM2, LDHA, MCT4, GLUT1, GPR81 protein or mRNA expression, and morphological abnormalities of trophoblast cells improved significantly in the RSA mice after dydrogesterone or different doses of STW treatment (P < 0.05). Conclusion: STW can promote aerobic glycolysis in trophoblast cells of RSA mouse embryos, thereby improving the microenvironment of the maternal-fetal interface and enhancing embryo implantation.

Morroniside Protects Human Granulosa Cells against H2O2-Induced Oxidative Damage by Regulating the Nrf2 and MAPK Signaling Pathways.

Morroniside is the main ingredient of Cornus officinalis and has a variety of biological activities including antioxidative effects. Ovarian granulosa cells (GCs) are responsible for regulating the development and atresia of follicles, which are susceptible to oxidative stress. In this study, we determined whether morroniside can inhibit the oxidative stress of GCs induced by hydrogen peroxide (H2O2), leading to improved oocyte quality. The oxidative damage and apoptosis of ovarian GCs cultured in vitro were induced by the addition of H2O2. After pretreatment with morroniside, the levels of ROS, MDA, and 8-OHdG in ovarian GCs were significantly decreased. Morroniside significantly upregulated p-Nrf2 and promoted the nuclear translocation of Nrf2, which transcriptionally activated antioxidant SOD and NQO1. In addition, morroniside significantly regulated the levels of apoptosis-related proteins Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3 via the p38 and JNK pathways. These results suggest that morroniside can reduce the oxidative damage and apoptosis of ovarian GCs induced by H2O2.

Clinical efficacy of Wenjing decoction in the treatment of ovulatory disorder infertility: A systematic review and meta-analysis.

OBJECTIVES: Wenjing decoction (WJD) was widely used in the treatment for ovulatory disorder infertility (ODI) in China, while its efficacy was not clearly known. In this study, we evaluated the clinical efficacy of WJD by meta-analysis. METHODS: Eight electronic databases including Cochrane Library, PubMed, Embase, Web of Science, China National Knowledge Infrastructure, WanFang Data, VIP Database, and China Biology Medicine were searched for randomized controlled trials (RCTs) published from the inception of each database to July 1, 2021, of which the interventions involve WJD and clomiphene. Outcomes included clinical efficacy rate, pregnancy rate, ovulation rate, dominant follicle diameter, endometrial thickness, estradiol, follicle-stimulating hormone, and luteinizing hormone. Meta-analysis and risk of bias were performed by RevMan 5.3 software. RESULTS: Eleven RCTs including 915 patients, of which 476 in the intervention group and 439 in the control group. Meta-analysis showed that WJD was better than clomiphene for patients with ODI in terms of clinical effective rate (odds ratio [OR] = 1.22, 95% confidence interval [CI]: 1.08-1.34), pregnancy rate (OR = 1.54, 95% CI: 1.15-2.07), ovulation rate (OR = 1.34, 95% CI: 1.07-1.67), endometrial thickness (mean difference [MD] = 1.50, 95% CI: 0.90-2.10), and dominant follicle diameter (MD = 1.85, 95% CI: 0.68-3.02). The estradiol level (MD = 91.0, 95% CI: 80.3-101.88) in patients taking WJD was significantly higher than those taking clomiphene, while the follicle-stimulating hormone level (MD = -0.93, 95% CI: -1.13 to -0.72) and the luteinizing hormone level (MD = -4.41, 95% CI: -4.80 to -4.03) in patients taking WJD was significantly lower than those taking clomiphene. Our results also indicated that WJD combined with clomiphene was better than clomiphene alone for patients with ODI in terms of pregnancy rate (OR = 1.79, 95% CI: 1.37-2.35). CONCLUSIONS: WJD may be effective in the treatment of patients with ODI. Due to the quality and quantity of literature, RCT with large sample size and high quality need to be performed to verify our conclusion.

N-Acetylcysteine improves oocyte quality through modulating the Nrf2 signaling pathway to ameliorate oxidative stress caused by repeated controlled ovarian hyperstimulation.

CONTEXT: N -acetyl-cysteine (NAC) is a potent antioxidant that can be used for many gynecological diseases such as polycystic ovary syndrome and endometriosis. Controlled ovarian hyperstimulation (COH) is a critical step in infertility treatment. Our previous clinical studies have shown that repeated COH led to oxidative stress in follicle fluid and ovarian granulosa cells. AIMS: In this study, we investigated whether NAC could inhibit oxidative stress in mice caused by repeated COH and improve the mitochondrial function of oocytes. METHODS: Female Institute of Cancer Research (ICR) mice were randomly assigned into three groups: normal group, model (repeated COH) group, NAC group. We examined the morphology, number and quality of mitochondria. The mechanism of regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) by NAC to ameliorate oxidative stress was also investigated. KEY RESULTS: Repeated COH caused oxidative damage in ovaries and oocytes and decreased oocyte quality, while NAC prevented oxidative damage and increased oocyte mitochondrial function. In in vitro experiments, it was verified that NAC can promote the nuclear translocation of Nrf2, which transcriptionally activates the expression of superoxide dismutase and glutathione peroxidase, which removed excessive reactive oxygen species that causes mitochondria damage. CONCLUSIONS: The results suggest that NAC raises mitochondrial function in oocytes and improves oocyte quality through decreasing oxidative stress in mice with repeated COH. The underlying mechanism is related to the regulation of the Nrf2 signaling pathway. IMPLICATION: This study provides a meaningful foundation for the future clinical application of NAC during repeated COH.

Xiaoyao powder improves endometrial receptivity via VEGFR-2-mediated angiogenesis through the activation of the JNK and P38 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoyao powder (XYP) is a traditional Chinese medicine formula which has wide scope of indications related to liver stagnation, reconcile qi and blood in TCM syndrome. Infertility can induce similar symptoms and signs to the clinical features of liver stagnation syndrome, the treatment of infertility by soothing the liver is obvious. XYP can increase the clinical pregnancy rate, follicle development, oocyte quality and improve endometrial receptivity. However, its underlying pharmacological mechanism of improving endometrial receptivity is unclear. AIM OF THE STUDY: The aim of the study was to investigate the effect of XYP on pregnancy rates and endometrial angiogenesis, to determine the potent mechanism in association with the pro-angiogenic behavior which closely related to improving endometrial receptivity. MATERIALS AND METHODS: We established an animal model exhibiting decreasing endometrial receptivity by controlled ovarian hyperstimulation and a human endometrial microvascular endothelial cell (HEMEC) model. Endometrial morphology was observed by hematoxylin-eosin staining and Scanning electron microscopy. Western blot and qRT-PCR analysis were used to detect expression of PCNA, Cyclin D1, MMP9 and MAPK signaling pathway. Scratch-wound assay and tube formation assay were used to observe HEMEC migration and tubulogenesis. RESULTS: The results demonstrated that XYP pretreatment could improve endometrial receptivity, which leads to high pregnancy rates. In the endometrium, XYP facilitated angiogenesis by promoting tube formation. XYP could enhance HEMEC proliferation and migration induced by VEGF, which were observed by the microscope and Scratch-wound assays. XYP promoted HEMEC proliferation and migration via the p38 and JNK MAPK signaling pathways. CONCLUSION: XYP promotes HEMEC proliferation and migration via the P38 and the JNK MAPK signaling pathways, which contribute to the endometrial angiogenesis mediated by VEGFR-2 that is favorable for endometrial receptivity. We firstly elucidated the molecular mechanisms by which XYP improved endometrial receptivity by promoting angiogenesis.

Effects of Bushen-Tiaojing-Fang on the pregnancy outcomes of infertile patients with repeated controlled ovarian stimulation.

Bushen-Tiaojing-Fang (BSTJF) is commonly used to treat infertility. This study investigated the effects of BSTJF on the pregnancy outcomes of patients with repeated controlled ovarian stimulation (COS), on mitochondrial function, and on oxidative stress in ovarian granulosa cells (GCs) and follicular fluid (FF). The samples and clinical data of 97 patients, including 35 in the control group, 29 in the placebo group and 33 in the BSTJF group, were collected for this study. The mitochondrial ultrastructure, ATP content, mitochondrial DNA (mtDNA) number, 8-hydroxy-2-deoxyguanosine (8-OHdG), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase (GSH-Px) activity levels, and mRNA expression levels of Mn-SOD, GSH-Px, and nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) were analyzed. The high-grade embryo (P < 0.001), implantation (P = 0.033), and clinical pregnancy (P = 0.031) rates, as well as the ATP content (P = 0.014), mtDNA number (P = 0.035), GSH-Px activity (P = 0.004 in GCs and P = 0.008 in FF) and mRNA expression levels (P = 0.019), were significantly lower in the placebo group than in the control group, whereas the 8-OHdG content was significantly (P = 0.006 in FF) higher in the placebo group than in the control group. Compared with those in the placebo group, the high-grade embryo rate (P = 0.007), antioxidant enzyme activity (P = 0.037 and 0.036 in Mn-SOD; P = 0.047 and 0.030 in GSH-Px) and mRNA level (P < 0.001 in Nrf2, P = 0.039 in Mn-SOD and P = 0.002 in GSH-Px) were significantly higher in the BSTJF group, as were changes in mitochondrial ultrastructure, ATP (P = 0.040) and mtDNA number (P = 0.013). In conclusion, BSTJF can improve oxidative stress in patients with repeated COS and pregnancy outcomes.

Effects of multicycle gonadotropin-releasing hormone antagonist protocols on oxidative stress of follicular fluid and ovarian granulosa cells.

The effect of repeated multicycle gonadotropin-releasing hormone antagonist (GnRH-ant) protocols on oxidative stress (OS) in follicular fluid (FF) and ovarian granulosa cells (GCs) remains unclear. This study investigated the effects of repeated multicycle GnRH-ant protocols on OS markers of FF and ovarian GCs. A total of 145 patients were enrolled and divided into four groups: 1 cycle group (n = 42), 2 cycles group (n = 37), 3 cycles group (n = 45), and 4-5 cycles group (n = 21). The FF and ovarian GCs of the patients were collected on the day of last oocyte retrieval and the levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were tested by ELISA. The results showed that the serum estradiol levels on hCG injection day in the 3 and 4-5 cycles were significantly (P < 0.05) lower than in the 1 and 2 cycles. The number of retrieved oocytes (12.1 ± 3.3 in cycle 1, 11.7 ± 3.1 in cycle 2, 10.4 ± 2.4 in cycle 3, and 9.4 ± 2.4 in cycles 4-5), embryos with two pronuclei (7.6 ± 3.0 in cycle 1, 7.0 ± 2.5 in cycle 2, 6.2 ± 2.6 in cycle 3, and 5.5 ± 2.1 in cycles 4-5), and the rates of high-quality embryos (52.2% in cycle 1, 47.9% in cycle 2, 38.6% in cycle 3, and 36.5% in cycles 4-5), implantation (35.4% in cycle 1, 32.4% in cycle 2, 23.8% in cycle 3, and 22.9% in cycles 4-5) and clinical pregnancy (50.0% in cycle 1, 43.2% in cycle 2, 33.3% in cycle 3, and 23.8% in cycles 4-5) in cycles 3 and 4-5 were significantly (P < 0.05) lower than those in cycles 1 and 2. Compared with 1 and 2 cycles, the 8-OHdG and SOD were significantly increased in the 3-5 cycles, while the CAT and GSH-Px levels were significantly decreased. Together, this study reveals repeated COS with the use of GnRH-ant protocols results in OS and changes the follicle microenvironment of FF and GCs, possibly leading to poor IVF outcomes in patients with 3-5 cycles of COS.

FOXD3­AS1/miR­128­3p/LIMK1 axis regulates cervical cancer progression.

Long non­coding RNA forkhead box D3 antisense RNA 1 (FOXD3­AS1) functions as an oncogenic regulator in several types of cancer, including breast cancer, glioma and cervical cancer. However, the effects and mechanisms underlying FOXD3­AS1 in cervical cancer (CC) are not completely understood. The present study aimed to investigate the biological functions and potential molecular mechanisms underlying FOXD3­AS1 in CC progression. Reverse transcription­quantitative PCR was performed to detect FOXD3­AS1, microRNA (miR)­128­3p and LIM domain kinase 1 (LIMK1) expression levels in CC tissues and cells. Immunohistochemical staining and western blotting were conducted to assess LIMK1 protein expression levels in CC tissues and cells, respectively. Cell Counting Kit­8 and BrdU assays were used to determine the role of FOXD3­AS1 in regulating cell proliferation. CC cell migration and invasion were assessed by performing Transwell assays. Dual­luciferase reporter assays were conducted to verify the binding between miR­128­3p and FOXD3­AS1. FOXD3­AS1 expression was significantly increased in CC tissues and cell lines compared with adjacent healthy tissues and normal cervical epithelial cells, respectively. High FOXD3­AS1 expression was significantly associated with poor differentiation of tumor tissues, increased tumor size and positive lymph node metastasis. FOXD3­AS1 overexpression significantly increased CC cell proliferation, migration and invasion compared with the negative control (NC) group, whereas FOXD3­AS1 knockdown resulted in the opposite effects compared with the small interfering RNA­NC group. Moreover, the results demonstrated that FOXD3­AS1 targeted and negatively regulated miR­128­3p, which indirectly upregulated LIMK1 expression. Therefore, the present study demonstrated that FOXD3­AS1 upregulated LIMK1 expression via competitively sponging miR­128­3p in CC cells, promoting CC progression.

Bushen Tiaojing (II and III) Decoctions Activate MAPK14 and MAPK3/1 to Promote the Expression of Cumulus Expansion-Related Factors in Mice.

BACKGROUND: Bushen Tiaojing Decoctions (BSTJ-II-D and BSTJ-III-D) are used to assist pregnancy in clinical practice. In this study, we explored the ability of sequential administration of BSTJ-II-D and BSTJ-III-D to promote cumulus cell (CC) expansion and its underlying mechanisms in controlled ovarian hyperstimulation (COH) mice. METHODS: Kunming mice were randomly divided into three groups. The normal group was injected intraperitoneally with saline, and distilled water was administered orally by gavage. As the COH model, mice were injected with GnRHa, eCG, and hCG. Subsequently, the BSTJD group received BSTJ-II-D and BSTJ-III-D orally by gavage, while the control group received distilled water. We evaluated CC expansion and oocyte first polar body (PB1) extrusion under a stereomicroscope. Serum levels of follicle-stimulating hormone (FSH) were detected by radioimmunoassay. The expression of the CC expansion-related factors PTX3 and PTGS2 was detected by immunofluorescence, western blot, and quantitative real-time-polymerase chain reaction analyses (qRT-PCR). Expression of p-MAPK14, p-MAPK3/1, MAPK14, and MAPK3/1 was detected by western blot analysis. RESULTS: Sequential administration of BSTJ-II-D and BSTJ-III-D promoted cumulus expansion and oocyte PB1 extrusion and upregulated PTX3 and PTGS2 expression at the mRNA and protein levels. Furthermore, the levels of p-MAPK14/MAPK14, p-MAPK3/1/MAPK3/1 proteins, and serum FSH in the BSTJD group were higher than those in the normal and control groups. CONCLUSIONS: Sequential administration of BSTJ-II-D and BSTJ-III-D promotes cumulus expansion and oocyte maturation in COH mice by increasing FSH expression and activating the MAPK14 and MAPK3/1 signalling pathways, thereby increasing expression of PTX3 and PTGS2.

[Expert consensus statement on Kangfu Xiaoyan Suppository in treatment of pelvic inflammatory in clinical practice].

Kangfu Xiaoyan Suppository is widely used in the treatment of gynecological inflammatory diseases. Long-term clinical application and a certain amount of research evidences show that Kangfu Xiaoyan Suppository can alleviate the clinical symptoms of pelvic inflammatory diseases,reduce the recurrence rate,and relieve sequelae,with a better safety and economic characteristics. As a type of nationally protected traditional Chinese medicine and type B medicine included in medical insurance,it has been selected as a Chinese patent medicine for rectal administration. It was included in the Guidelines for diagnosis and treatment of common gynecological diseases of traditional Chinese medicine published by the Chinese Academy of Traditional Chinese Medicine in 2012,the Pelvic inflammatory diseases diagnosis and treatment guidelines issued by the Infectious Diseases Collaborative Group of the Obstetrics and Gynecology Branch of the Chinese Medical Association in 2014,and the group standard of Single use of traditional Chinese medicine/combined antibiot guidelines for clinical practice-pelvic inflammatory diseases of the Chinese Academy of Traditional Chinese Medicine in 2017. To further enhance clinicians' understanding of the drug and better guide its rational clinical use,experts from the field of gynecology of traditional Chinese and Western medicine were invited to develop and compile this expert consensus. This consensus takes full account of clinical evidences and expert clinical experience,and form recommendations for clinical problems based on evidences and consensus recommendations for clinical problems without evidence by nominal grouping method. The expert consensus is mainly formed in the consideration of six factors: quality of evidence,economy,efficacy,adverse reactions,patient acceptability and others. Based on clinical research evidences and expert experience,this consensus provides a preliminary reference for the clinical use of the drug in a concise and clear format. However,evidence-based support is still required in a large number of high-quality studies,and this consensus will be revised in the future according to new clinical problems and the update of evidence-based evidence in practical application.

[PM 2.5 and Emergency Calls for Health with Respiratory Diseases in Chengdu in 2017].

OBJECTIVE: To determine the association between daily particulate matter 2.5 (PM 2.5) mass and emergency calls for help with respiratory diseases. METHODS: Semi-parametric generalized additive model was established to determine the association between daily PM 2.5 and emergency calls for help with respiratory diseases in 2017 in Chengdu, after adjustments for time trend and variations in the days of the week and weather conditions. RESULTS: In 2017, a total of 9 309 emergency calls for help with respiratory diseases were recorded in Chengdu: on average 26 calls a day. Over the year, Chengdu reported a mean PM 2.5 mass concentration of 53.6 µg/m 3, an average temperature of 16.6 ℃, and an average relative humidity of 81.2%. The single pollutant model with lag time effect showed that a 10 µg/m 3 increase in PM 2.5 was associated with an increase of 1.26% (95% confidence interval ( CI) 0.56%-1.97%) emergency calls for help with respiratory diseases. The exposure-response was almost in a direct line. The dual pollutant model found that O 3 8-hour sliding average (O 3-8 h) enhanced the effect of PM 2.5 on emergency calls for help with respiratory diseases. CONCLUSION: Outdoor PM 2.5 is a significant predictor of emergency calls for help with respiratory diseases in Chengdu.

Bu Shen Zhu Yun Decoction Improves Endometrial Receptivity via VEGFR-2-Mediated Angiogenesis.

Vascular endothelial growth factor receptor-2 (VEGFR-2) regulates the mitogen-activated protein kinase (MAPK) signaling pathway and plays an important role in angiogenesis. Bu Shen Zhu Yun decoction (BSZYD) can improve endometrial receptivity and embryo implantation rates in patients undergoing in vitro fertilization. However, whether BSZYD improves endometrial receptivity via angiogenesis remains unclear. Here, we investigated the effects of BSZYD on the proliferation, migration, and angiogenesis of human endometrial microvascular endothelial cells (HEMECs) and found that BSZYD upregulated the expression of cyclin D1, matrix metalloproteinase 9 (MMP9), and proliferating cell nuclear antigen (PCNA) in HEMECs. Cell Counting Kit 8 assay, scratch-wound assay, and Tube Formation Assay results showed that BSZYD promoted the proliferation, migration, and angiogenesis of HEMECs. Western blot analysis results revealed the activation of the MAPK signaling pathway by BSZYD through the upregulation of VEGF and VEGFR-2 expression. Together, these findings highlight the novel mechanism underlying BSZYD-mediated improvement in endometrial receptivity through the MAPK signaling pathway.

[Effect of GnRHa controlled ovarian hyperstimulation on mouse embryo implantation and its mechanism].

The purpose of the present study was to investigate the effects and underlying mechanism of gonadotropin-releasing hormone agonist (GnRHa) controlled ovarian hyperstimulation (COH) on embryo implantation in mice. Forty female Kunming mice aged 9 weeks were randomly divided into two groups (control and COH groups). The COH group received intraperitoneal (i.p.) injections of aminocyclin acetate (GnRHa), human menopausal gonadotropin (HMG) and human chorionic gonadotropin (hCG), while the control group was given equal amount of physiological saline by i.p. injection. One male mouse and two female mice were put into the same cage at 16:00 on the hCG injection day, and on the fourth day of pregnancy, 10 mice from each group were killed. The levels of serum estradiol (E2) and progesterone (P) were measured by radioimmunoassay; HE staining was used to observe the morphology of ovarian and endometrial tissues. The protein expression levels of endometrial leukemia inhibitory factor (LIF), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and glycodelin A were detected by Western blot and immunohistochemistry. Ten mice from each group were sacrificed on the eighth day of pregnancy, and the status of the uterus and the average number of blastocysts were observed. The results showed that, compared with control group, the serum E2 level in COH group was significantly decreased (P < 0.05), while the P level was increased significantly (P < 0.05); the ovarian follicles at different developmental stages were rare, corpus lutea (CL) were visible and multiple, the endometrium was thinned, and the number of endometrial glands was reduced (P < 0.05); the contents of LIF, p-STAT3, HB-EGF and glycodelin A in the endometrium were decreased significantly (P < 0.05) on the fourth day of pregnancy; mouse blastocysts developed slowly and were decreased in number on the eighth day of pregnancy (P < 0.05). The above results suggest that GnRHa COH can affect embryo implantation in mice. The mechanism may be related to the imbalance of gonadal hormone, the changes in the structure of the endometrium and the expressions of LIF, p-STAT3, HB-EGF and glycodelin A in the implantation stage, which may lead to the decrease of endometrial receptivity and the abnormal dialogue between the embryo and the uterus.