Effect of dominant follicle status at the time of retrieval on the clinical outcomes in natural cycle IVF combined with immature oocyte treatment.

OBJECTIVE: It is commonly believed that the oocytes from small follicles are unhealthy when a dominant follicle (DF) is recruited in the ovaries, especially when the DF is ovulated. This study aims to confirm whether the presence or ovulation of DF at the time of retrieval affects the clinical outcome of the natural cycle IVF with in vitro maturation (NC-IVF/M) treatment. METHODS: Data were collected from 446 women with regular menstrual cycle and 536 retrieval cycles using NC-IVF/M treatment. The cycles were divided into three groups based on the results of the oocyte retrieval cycle. Group A covers the collection of oocytes from the DF and small follicles; Group B incorporates failed oocyte retrieval from DF and then the oocytes are retrieved only from small follicles; and Group C includes the retrieval of oocytes only from small follicles accompanied with an ovulated DF. Furthermore, Group B and C have subgroups to include whether in vivo matured oocytes were obtained from small follicles. Following aspiration of DF and small follicles, mature oocytes were inseminated on the date of retrieval by intracytoplasmic sperm injection (ICSI) and the immature oocytes were matured in vitro. If the immature oocytes were matured in vitro, they were inseminated using ICSI, and then the embryos obtained from in vivo and in vitro matured oocytes were transferred accordingly. RESULTS: The oocytes from DF were successfully retrieved in 445 cycles (83.0%), failed to be retrieved in 54 cycles (10.1%) and ovulated in 37 cycles (6.9%). In Group A, an average of 2.0 ± 1.7 mature oocytes were retrieved, which was significantly higher than the average of Group B, with 1.3 ± 1.3 matured oocytes and Group C, with an average of 1.1 ± 1.5 matured oocytes (P < 0.01). However, the average number of immature oocytes retrieved from each group show no difference among the three groups. There was no significant difference in maturation rates of immature oocytes, fertilization rates among the three groups. The clinical pregnancy rate per transfer cycle is 34.5%, 34.6% and 25.7% in Group A, B and C, respectively. No significant differences were observed in embryonic development and implantation capacity in Group B and C in comparison to Group A. And there was no significant difference in clinical pregnancy, implantation, live birth and miscarriage rates among the three groups. No significant differences were observed in the developmental and implantation capacity according to with or without in vivo matured oocytes were retrieved in Group B and Group C. CONCLUSION: The presence or ovulation of the dominant follicle from the ovaries does not significantly influence the developmental and implantation capacity of immature oocytes retrieved from small follicles, suggesting that NC-IVF/M is a promising treatment option for women without ovarian stimulation.

Effect of Huanglian Decoction on the Intestinal Microbiome in Stress Ulcer (SU) Mice.

BACKGROUND: Stress ulcer (SU) is a serious gastrointestinal mucosal lesion under acute stress. Huanglian decoction is a famous traditional Chinese medicine prescription, which has been used to treat digestive system diseases for thousands of years. Many clinical cases have proved that Huanglian decoction has a good effect on SU. Some studies have shown that the intestinal bacteria will be changed accordingly when the gastrointestinal mucosa is damaged. However, there are few published reports on the effect of the intestinal microbiome with SU mice that were treated by Huanglian decoction. In this study, we investigated the effect of the fecal microbiome in mice with SU by the 16S rDNA sequencing technology. METHODS: Male KM mice were induced by cold-restraint stress except for the normal control group and then treated by Huanglian decoction (Group HD) and Esomeprazole magnesium solution (Group ES) separately for 7 days. 16S rDNA sequencing technology analysis was applied to evaluate the changes of bacterial flora in mice feces. And, histopathological methods and molecular biological detection methods were also performed. RESULTS: Huanglian decoction could help to repair the gastric mucosal injury and regulate the relative content of TNF-α and IL-1ß. Moreover, Huanglian decoction could increase the relative abundance of intestinal probiotics in the intestine of mice with SU, especially in Verrucomicrobiae and Akkermansia. CONCLUSIONS: Huanglian decoction might effectively promote the repair of gastrointestinal mucosal injury and regulate the number and species of intestinal bacteria to maintain the stability of gastrointestinal microecology.

[Nuclear magnetic resonance-based metabolomics revealed metabolite profiles participate in intervention effect of refined moxibustion in gastric ulcer model rats].

OBJECTIVE: To investigate the effect of refined moxibustion on expression of gastric mucosal epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), and changes of metabolite profiles in gastric ulcer (GU) rats, so as to analyze its mechanism underlying improvement of GU. METHODS: Male SD rats were randomized into control, model, acupoint moxibustion groups (n=6 per group). The GU model was induced by cold-restraint stress. The ignited refined moxa was applied to bilateral "Liangmen" (ST21) and "Zusanli" (ST36) for 3 cones/acupoint, once daily for 7 days. Then, we employed 1H NMR-based metabolomics approach to analyze the metabolic profiles of serum and stomach tissue samples. The conventional histopathological changes of the gastric mucosa were observed by H.E. stain and the expressions of EGFR and VEGF in the gastric mucosa were detected by immunohistochemistry. RESULTS: Compared to the control group, the expression levels of EGFR and VEGF were significantly increased in the model group (P<0.01, P<0.05), and further notably up-regulated in the acupoint moxibustion group (P<0.001, P<0.01). Results of H.E. staining showed damage of the folds of gastric mucosa, disordered arrangement of the glands, infiltration of inflammatory cells and unclear structure of gastric mucosa in the model group, which was relatively milder in the acupoint moxibustion group. 1H-NMR technical analysis showed that in comparison with the control group, 11 and 11 metabolites in the stomach extract and plasma were increased, 10 in the gastric tissue and 3 in the plasma were decreased in the GU model group; while in comparison with the model group, 17 differently expressed metabolites in the gastric extract and 10 metabolites in the plasma restored to their levels of control group after the acupoint moxibustion intervention. These metabolites participate in 12 metabolic pathways including glycine, serine and threonine metabolism, glutathione metabolism, glycine metabolism, alanine, aspartic acid and glutamic acid metabolism, purine metabolism, glyoxylic acid and digarboxylic acid metabolism, biosynthesis of aminoacyl-tRNA, amino sugar and nucleotide sugar metabolism, cysteine and methionine metabolism, citrate cycle, pyruvate metabolism, and the mutual conversion of pentose and glucuronate，suggesting their involvement in moxibustion-induced improvement of GU. CONCLUSION: Refined moxibustion at ST21 and ST36 can up-regulate the expression of EGFR and VEGF in the gastric mucosa and lessen gastric mucosal injury, which may be related to its effects in reducing GU-induced metabolic disorders, including sugar, purine, amino acid, and phospholipid metabolism and antioxidant defense system.

[Effects of manual loading on calcitonin gene-related peptide and nerve growth factor in rats with chronic low back pain].

OBJECTIVE: To observe the analgesic effect of manipulation loading on chronic low back pain (CLBP) model rats and the expression of inflammatory factors in psoas major muscle tissue, and to explore the improvement of manipulation on local inflammatory microenvironment. METHODS: Thirty two SPF male SD rats weighing 340-360g were randomly divided into blank group, sham operation group, chronic low back pain model group and treatment group, with 8 rats in each group. In the model group, L4-L6 lumbar vertebrae were implanted with external link fixation system (ELFS). After implantation of ELFS, the treatment group received manualintervention with 5N force and 2Hz frequency on both sides of the spine, 15 min / time, once a day, for 14 consecutive days. Paw with drawl threshold (PWT) and paw withdrawl latency (PWL) was measured before modeling and on the 1st, 3rd, 7th, 10th and 14th day after intervention. At the end of the treatment cycle, the concentrations of calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) in psoas muscle were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: There was no significant difference in PWT and PWL between the blank group and the sham operation group after modeling (P>0.05);after modeling, PWT and PWL in the CLBP model group and the treatment group were significantly decreased(P<0.01);PWT in the treatment group was not significantly improved than that in the CLBP model group on the 1st and 3rd day after manual loading(P>0.05);on the 7th day after manual loading, the pain threshold value in the treatment group was higher than that in the CLBP model group, but there was no significant difference There was no significant difference between the two groups (P=0.056>0.05). On the 10th and 14th day of treatment, the mechanical pain threshold of the treatment group began to rise, and it was statistically significant compared with CLBP model rats (P<0.05, P< 0.01);on the 1st and 3rd day after manual treatment, the PWL of the treatment group was not significantly improved compared with CLBP model group (P>0.05);on the 7th day, the PWL of the treatment group was significantly higher than that of CLBP model group, there was statistical significance (P=0.016<0.05). Manual loading improved thermal hyperalgesia in CLBP rats until the end of the experiment. The contents of CGRP and NGF in psoas major muscle of CLBP model group were higher than those of blank group and sham operation group (P<0.01). After treatment, the contents of CGRP and NGF decreased significantly(P<0.01). CONCLUSION: Local massage loading has analgesic effect on CLBP rats, at the same time, it can inhibit the content of CGRP and NGF in psoas muscle tissue of CLBP rats, and improve the local inflammatory microenvironment.

[Oral acute toxicity of (+)-usnic acid in mice and its cytotoxicity in rat cardiac fibroblasts].

OBJECTIVE: To observe the oral acute toxicity of of (+)-usnic acid in mice and assess its cytotoxicity in rat cardiac fibroblasts. METHODS: The mice with acute poisoning of (+)-usnic acid at different doses by oral administration were observed for toxic manifestations, and the LD(50) was determined. The survival time and survival rate of the mice receiving different doses of (+)-usnic acid were observed. Cultured rat cardiac fibroblasts were inoculated with different concentrations of (+)-usnic acid, and the cell growth inhibition rate was estimated and the IC(50) determined using MTT assay. RESULTS: Higher dose of (+)-usnic acid resulted in more obvious symptoms of poisoning and shorter survival time of the mice. The LD(50) of (+)-usnic acid in mice by oral administration was 388 mg/kg. The manifestations of poisoning such as apathism, pilomotor, chill, dyspnea, torpidity and anorexia was observed. Rat cardiac fibroblasts incubated with (+)-usnic acid showed obvious growth inhibition, which was positively correlated to the dose of (+)-usnic acid, and high dose of (+)-usnic acid caused severe cell injuries. The IC(50) of (+)-usnic acid in rat cardiac fibroblasts was 322 microg/ml. CONCLUSION: (+)-usnic acid is a natural compound of low toxicity in mice, and low to medium dose of (+)-usnic acid dose not produce obvious cytotoxicity.

Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice.

To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.

[Construction of multi-epitope DNA vaccine for Toxoplasma gondii and the study on protective immunity response in mice].

AIM: To construct multi-epitope DNA vaccine for Toxoplasma gondii and study its protective immunity response. METHODS: The gene encoding six polypeptides of T. gondii, which consists of plenty of T and B epitopes, was cloned into the eucaryotic expression vector pcDNA3.1(+). BALB/c mice were vaccinated by this multi-epitope based DNA vaccine (intramuscular needle injection). The specific antibody and T cell proliferation were determined. Meanwhile, the DNA-vaccinated mice were challenged with a lethal dose of T. gondii tachyzoites for further observation. RESULTS: The eukaryotic expression plasmid (pcDNA3.1/T-ME) encoding plenty of T. gondii epitopes was constructed successfully. pcDNA3.1/T-ME immunization induced T. gondii specific humoral and cellular immunity in mice. The mice immunized with pcDNA3.1/T-ME survived significantly longer than the mice in control after challenged by T. gondii RH strain infection. CONCLUSION: The multi-epitope DNA vaccine can induce the protective immunity against T. gondii infection effectively in vivo, which is a potential strategy to control T. gondii infection.

[In vitro effect of (+)-usnic acid on Toxoplasma gondii tachyzoites].

OBJECTIVE: To explore the effect of usnic acid on Toxoplasma gondii tachyzoites in vitro. METHODS: There are four groups named as (+)-usnic acid group, acetylspiramycin group, DMSO group and normal saline group. Groups of (+)-usnic acid and acetylspiramycin were further divided into 4 subgroups with final concentration of 5, 10, 25, 50 microg/ml respectively. Normal saline group and DMSO group were respectively given equal volume normal saline and 1% DMSO. Each group have 15 parallel tubes with 1 ml (1 x 10(6)/ml) T. gondii tachyzoites aqueous suspension. At 1 h, 2h and 4 h after drug treatment, tachyzoites were counted by light microscope with 0.4% Trypan blue staining. Tachyzoites in aqueous suspension was collected, and washed 3 times by PBS solution. Normal mice were inoculated intraperitoneally and observed for three generations. The cultivated rat cardiofibroblasts were then infected in vitro with T. gondii tachyzoites. At the same time, rat cardiomyocytes invasion by T. gondii tachyzoites was investigated. RESULTS: At 4 h treated by 10, 25 and 50 microg/ml (+)-usnic acid, 100% T. gondii tachyzoites were stained. Some tachyzoites were swelling, blunt or round in the two ends; and granules appeared in the cytoplasm, the nuclei were deep stained. The changes of tachyzoites in acetylspiramycin group were similar to (+)-usnic acid group, 100% T. gondii tachyzoites were stained in 50 microg/ml acetylspiramycin subgroup. In inoculation tests, mice died at 8th to 9th days in 5 microg/ml (+)-usnic acid subgroup and numerous tachyzoites were detected in ascites. However, most mice survived to be killed in the other (+)-usnic acid subgroups and the tachyzoites were not found in ascites. All mice in acetylespirmycin groups died at 6th to 8th days after inoculation and many tachyzoites or pseudocysts were observed in mice ascites. In infecting cell tests, the cultivated rat cardiofibroblasts were infected in vitro by the tachyzoites after treated with 5 microg/ml (+)-usnic acid for 4 h, and pseudocysts were formed in infected cells. It was negative in the other subgroups of (+)-usnic acid. But the cultivated rat cardiofibroblasts were infected to varying degree in acetylspiramycin groups, normal saline group and DMSO group. CONCLUSION: (+)-Usnic acid has a remarkable effect on T. gondii tachyzoites.

[Experimental study on the in vitro resistance to Toxoplasma gondii by murine lymphocyte].

AIM: To study the activities of in vitro resistance to the tachyzoite of Toxoplasma gondii by murine lymphocytes. METHODS: The rat's splenocyte culture method was used to observe the effects of lymphocytes themselves and lymphocytes together with Mandelic( MA) on the invasion and proliferation of T. gondii in lymphocytes. At the same time acetylspiramycin was used as positive control. RESULTS: As compared with other somatic cells, the lymphocytes invaded by T. gondii still inhibited and killed the toxoplasma organisms in the presence of immunity, the effect safety dose of MA on inhibition of the invasion of T. gondii was not notable while the inhibition of the proliferation of T. gondii in lymphocytes was remarkable. CONCLUSION: Cell-mediated immunity(CMI) was an important factor that host resists the T. gondii infection. So, we should pay attention to improving the organism's CMI and take proper medicine so as to enhance the effect of lymphocyte's resistance to T. gondii.