Enhancing Oocyte Quality in Aging Mice: Insights from Mesenchymal Stem Cell Therapy and FOXO3a Signaling Pathway Activation.

Ovarian aging reduced the quality of oocytes, resulting in age-related female infertility. It is reported that mesenchymal stem cells (MSCs) therapy can improve age-related ovarian function decline and the success rate of in vitro maturation (IVM) in assisted reproductive therapy. In order to investigate the effectiveness and mechanisms of MSCs to enhance oocyte quality of cumulus oocyte complexes (COCs) in advanced age, this study focus on the respective functional improvement of oocytes and granulosa cells (GCs) from aging mice and further to explore and verify the possible mechanisms. Here, we studied a popular but significant protein of follicular development, Forkhead box O-3a (FOXO3a), which is a transcription factor that mediates a variety of cellular processes, but the functions of which in regulating oocyte quality in MSCs therapy still remain inconclusive. In this study, the RNA-seq data of metaphase II (MII) oocytes and GCs isolated from COCs confirmed that, GCs of immature follicles show the most potential to be the targeted cells of bone marrow mesenchymal stem cells (BMSCs) by FOXO3a signaling pathway. Furthermore, we demonstrated the effectiveness of BMSCs co-culture with aging COCs to enhance oocyte quality and found its mechanism to function via ameliorating the biological function of GCs by alleviating FOXO3a levels. These results provide significant fundamental research on MSCs therapy on ovarian aging, as well as offering guidance for raising the success rate of assisted reproductive technology such IVM in clinical and non-clinical settings.

Evaluative effectiveness of follicular output rate, ovarian sensitivity index, and ovarian response prediction index for the ovarian reserve and response of low-prognosis patients according to the POSEIDON criteria: a retrospective study.

The aim was to explore the implications of follicular output rate (FORT), ovarian sensitivity index (OSI), ovarian response prediction index (ORPI), and follicle-to-oocyte index (FOI) in low-prognosis patients defined by POSEIDON criteria. In total, 4030 fresh in vitro fertilization (IVF) cycles from January 2013 to October 2021 were included in this retrospective cohort analysis and were categorized into four groups based on the POSEIDON criteria. The FORT between Groups 1 and 2 (0.61 ± 0.34 vs. 0.65 ± 0.35, P = 0.081) and Groups 3 and 4 (1.08 ± 0.82 vs. 1.09 ± 0.94, P = 0.899) were similar. The OSI in the order from the highest to the lowest were 3.01 ± 1.46 in Group 1, 2.28 ± 1.09 in Group 2, 1.54 ± 1.04 in Group 3, and 1.34 ± 0.96 in Group 4 (P < 0.001). The trend in the ORPI values was consistent with that in the OSI. FORT, OSI, ORPI, and FOI complemented each other and offered excellent effectiveness in reflecting ovarian reserve and response, but they were not good predictors of clinical pregnancy rate (CPR) from IVF.

Dietary Antimicrobial Peptides Improve Intestinal Function, Microbial Composition and Oxidative Stress Induced by Aeromonas hydrophila in Pengze Crucian Carp (Carassius auratus var. Pengze).

There is increasing evidence for the potential use of antimicrobial peptides as dietary supplements and antibiotic substitutes. In this study, we analyzed the differential effects of varying levels of antimicrobial peptides on the intestinal function and intestinal microbial and disease resistance of Pengze crucian carp. Approximately 630 experimental fishes were randomized in the control group (G0: 0 mg/kg) and in five groups supplemented with different doses of AMPs (G1: 100 mg/kg, G2: 200 mg/kg, G3: 400 mg/kg, G4: 800 mg/kg, and G5: 1600 mg/kg) and were fed for ten weeks. Three replicates per group of 35 fish were performed. The results showed that AMPs promoted intestinal villus development and increased intestinal muscular thickness (p < 0.05) and goblet cell abundance. The enzymatic activities of all groups supplemented with AMPs were effectively improved. AMP supplementation significantly enhanced the activities of antioxidant enzymes and digestive enzymes in the intestines of G3 animals (p < 0.05). Compared with G0 animals, AMP-supplemented animals regulated the expression of intestinal immune-related genes and exhibited significant differences in the G3 animal group (p < 0.05). The abundance of intestinal Firmicutes and Bacteroidetes increased in the AMP-supplemented groups, but the Firmicutes/Bacteroidetes ratio was lower than that in the G0 group. AMP supplementation also decreased the abundance of Fusobacterium while increasing the proportion of Actinobacteria (p < 0.05). After Aeromonas hydrophila infection, the expression levels of anti-inflammatory factors in the intestinal tract of G3 animals were significantly upregulated, and the level of the proinflammatory factor was decreased (p < 0.05). The intestinal Cetobacterium levels of G3 animals were significantly increased (p < 0.01), while the Proteobacteria levels were decreased, and the intestinal goblet cell proliferation was significantly lower than that of G0 animals (p < 0.05). This indicates that groups supplemented with AMPs have better disease resistance than the G0 group and can rapidly reduce the adverse effects caused by inflammatory response. Taken together, the present results suggest that AMP supplementation can improve intestinal function and intestinal microbial and pathogen resistance in Pengze crucian carp.

Size Effects of Microplastics on Embryos and Observation of Toxicity Kinetics in Larvae of Grass Carp (Ctenopharyngodon idella).

Microplastics have caused great concern in recent years. However, few studies have compared the toxicity of different sizes of microplastics in fishes, especially commercial fishes, which are more related to human health. In the present study, we revealed the effects of varying sizes of microplastics on grass carp embryos and larvae using scanning electron microscopy (SEM) and fluorescence imaging. Embryos were exposed to 80 nm and 8 µm microplastics at concentrations of 5, 15, and 45 mg/L. Toxicity kinetics of various sizes of fluorescent microplastics were analyzed through microscopic observation in the larvae. Results found that nanoplastics could not penetrate the embryo's chorionic membrane, instead they conglutinated or aggregated on the chorion. Our results are the first to explore the defense mechanisms of commercial fish embryos against microplastics. Larvae were prone to ingesting their own excrement, resulting in microplastic flocculants winding around their mouth. For the first time, it was found that excreted microplastics could be reconsumed by fish and reaccumulated in the oral cavity. Microplastics of a certain size (1 µm) could be accumulated in the nasal cavity. We speculate that the presence of a special groove structure in the nasal cavity of grass carp larvae may manage to seize the microplastics with a particular size. As far as we know, this is the first report of microplastics being found in the nasal passages of fish. Fluorescence images clearly recorded the toxicity kinetics of microplastics in herbivorous fish.

Pore morphology and fractal dimension of ash deposited in catalyst diesel particulate filter.

Diesel particle filter (DPF) has been widely acknowledged as the most effective way to mitigate particulate matter emitted from diesel engines. Over time, ash mainly derived from lubricating oil will deposit in DPF, showing negative influence to engine performance, fuel economy, service life of DPF, and so on. Recently, the investigation about DPF backpressure characteristics and DPF regeneration process considering ash has gained attention. As a porous material, ash will play a key role in the DPF permeability. Thus, the pore morphology and fractal dimension of ash derived from three kinds of lube are addressed in this work. The results show that the changing tendency of the micropore specific surface and pore volume is consistent with the ash content in lubricant oil, which is owing to the chemical interaction of Ca and S contained in the oil during the complex DPF regeneration. There is no significant changing tendency of the mesopore and macropore properties because of the heterogeneity and complexity of ash. According to the fractal analyses, the Avnir equation shows excellent predictive accuracy for the pore surface fractal dimension of ash, which reflects that the ash pore surfaces are irregular.

Regulated emission characteristics of in-use LNG and diesel semi-trailer towing vehicles under real driving conditions using PEMS.

On-road driving emissions of six liquefied natural gas (LNG) and diesel semi-trailer towing vehicles (STTVs) which met China Emission Standard IV and V were tested using Portable Emission Measurement System (PEMS) in northern China. Emission characteristics of these vehicles under real driving conditions were analyzed and proved that on-road emissions of heavy-duty vehicles (HDVs) were underestimated in the past. There were large differences among LNG and diesel vehicles, which also existed between China V vehicles and China IV vehicles. Emission factors showed the highest level under real driving conditions, which probably be caused by frequent acceleration, deceleration, and start-stop. NOx emission factors ranged from 2.855 to 20.939 g/km based on distance-traveled and 6.719-90.557 g/kg based on fuel consumption during whole tests, which were much higher than previous researches on chassis dynamometer. It was inferred from tests that the fuel consumption rate of the test vehicles had a strong correlation with NOx emission, and the exhaust temperature also affected the efficiency of Selected Catalytic Reduction (SCR) after-treatment system, thus changing the NOx emission greatly. THC emission factors of LNG vehicles were 2.012-10.636 g/km, which were much higher than that of diesel vehicles (0.029-0.185 g/km). Unburned CH4 may be an important reason for this phenomenon. Further on-road emission tests, especially CH4 emission test should be carried out in subsequent research. In addition, the Particulate Number (PN) emission factors of diesel vehicles were at a very high level during whole tests, and Diesel Particulate Filter (DPF) should be installed to reduce PN emission.

Effects of Metformin Treatment on Soluble Leptin Receptor Levels in Women with Polycystic Ovary Syndrome.

The effects of metformin treatment on soluble leptin receptor (sOB-R) levels in women with polycystic ovary syndrome (PCOS) were investigated. This prospective and open-label study was conducted by the Department of Obstetrics & Gynecology at Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, China. Fifty-five women with PCOS and insulin resistance (IR) were treated with metformin for 6 months. According to body mass index (BMI), the patients were divided into two groups: lean PCOS group (BMI <23 kg/m2, n=34) and overweight or obese PCOS group (BMI ≥23 kg/m2, n=21). Before and after treatment, serum luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), androstenedione (A), dehydroepiandrosterone sulfate (DHEAS), insulin and sOB-R levels were determined. Thirty-one BMI-matched ovulatory women served as controls. The results showed: (1) The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), androgen levels and hirsutism scores were higher, and sOB-R levels were lower in PCOS groups than in control group. A subgroup analysis of lean and overweight or obese PCOS patients revealed there was significant difference in sOB-R level between lean PCOS group and overweight or obese PCOS group. There were no significant differences in anthropometric parameters between lean PCOS patients and BMI-matched controls. However, sOB-R level was significantly lower in lean PCOS women than in controls. (2) There was no correlation between sOB-R level and BMI, waist and hip circumference, total testosterone, androstendione, DHEAS, LH or hirsutism scores in PCOS patients, but there was a significant negative correlation between sOB-R and HOMA-IR. (3) After treatment with metformin for 6 months, serum insulin levels decreased, and sOB-R levels increased significantly (P<0.01). It was suggested that considering low sOB-R levels supposedly compensate diminished leptin action, PCOS per se might cause leptin resistance. It is likely that reduction of hyperinsulinemia produced by metformin effectively improves the sOB-R levels in PCOS.

Autophagy contributes to hypoxia-induced epithelial to mesenchymal transition of endometrial epithelial cells in endometriosis.

Endometriosis is a benign gynecologic disorder, and presents with malignant characteristics, such as migration and invasion. Hypoxia has been implicated in triggering epithelial-mesenchymal transition (EMT). Hypoxia is also known to induce autophagy. However, the relationship between autophagy and EMT under hypoxia conditions in endometriosis remains unknown. In the present study, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), microtubule associated protein light chain 3 (LC3), and mesenchymal cell marker vimentin was significantly higher in ectopic endometrium from patients with endometriosis, along with decreased expression of epithelial cell marker E-cadherin. After hypoxia treatment, endometrial epithelial cells exhibited enhanced migration and invasion abilities, as well as promoted autophagy and the EMT phenotype. Our analyses also show that HIF-1α was responsible for induction of autophagy. Moreover, inhibition of autophagy by chemical or genetic approaches suppressed hypoxia triggered EMT and reduced cell migration and invasion. Collectively, our findings identify that autophagy is critical for the migration and invasion of endometrial cells through the induction of EMT and indicate that inhibition of autophagy may be a novel useful strategy in the treatment of endometriosis.

Complement component Bf/C2b gene mediates immune responses against Aeromonas hydrophila in grass carp Ctenopharyngodon idella.

The complement system is a significant component of innate immunity. Here, we identified a Bf/C2 homolog (gcBf/C2b) in grass carp. gcBf/C2b shares a high similarity with Bf/C2b counterparts in other teleosts. gcBf/C2b transcription was widely distributed in different tissues, induced by Aeromonas hydrophila in vivo and in vitro, and affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcBf/C2b, transcript levels of all components except gcC5 were significantly enhanced, and gcBf/C2b, gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after A. hydrophila challenge or stimulation with bacterial pathogen-associated molecular patterns (PAMPs). However, gcBf/C2b in interference cells down-regulated the transcript levels after A. hydrophila challenge, and gcBf/C2b induced NF-κB signaling. These findings indicate the vital role of gcBf/C2b in innate immunity in grass carp.

Mannose-binding lectin and its roles in immune responses in grass carp (Ctenopharyngodon idella) against Aeromonas hydrophila.

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity via four pathways. Mannose-binding lectin (MBL), the initiating molecule of the lectin pathway, plays a significant role in the innate immune system in mammals and fish. Herein, we identified an MBL homolog (gcMBL) in grass carp (Ctenopharyngodon idella). The full-length 948 bp gcMBL cDNA includes a 741 bp open reading frame encoding a 246 amino acid protein with a signal peptide, collagen triple helix repeat domain, and a C-type lectin-like/link domain. The gcMBL protein shares low similarity with MBL counterparts in other species, and is most closely related to Cyprinus carpio MBL. Transcription of gcMBL was widely distributed in different tissues, and was induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMBL was also affected by LPS and flagellin stimulation in vitro. In cells over-expressing gcMBL, transcripts of almost all components except gcC5 were up-regulated, and gcMBL, gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR and gcITGß-2 were significantly up-regulated following exposure to A. hydrophila or stimulation by bacterial PAMPs. Meanwhile, gcMBL deficiency achieved by RNAi down-regulated transcript levels following A. hydrophila challenge, and gcMBL induced NF-κB signalling. These findings indicate a vital role of gcMBL in innate immunity in grass carp.

Mannan-binding lectin-associated serine protease-1 (MASP-1) mediates immune responses against Aeromonas hydrophila in vitro and in vivo in grass carp.

The mannan-binding lectin-associated serine protease-1 (MASP-1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP-1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP-1 homolog (gcMASP-1) in the grass carp (Ctenopharyngodon idella). The full-length 3308-bp gcMASP-1 cDNA includes a 2160-bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor-like, complement control protein, and trypsin-like domains. gcMASP-1 shares a high similarity with MASP-1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP-1 and Sinocyclocheilus anshuiensis MASP-1. Transcription of gcMASP-1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP-1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcMASP-1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after exposure to A. hydrophila; gcMASP-1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP-1 activated NF-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella.

Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila.

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens.

[Optimization of electroporation parameters for ctenopharyngodon idellus kidney cells and transient expression of grass carp reovirus NS26 protein].

In this study, pEGFP-N1 was chosen as the reporter plasmid and transferred into Ctenopharyngodon idellus kidney (CIK) cells by electroporation, and the optimal electroporation conditions were determined by testing the transfection efficiency with different voltages, pulse times, plasmid amounts, and numbers of shocks. The results showed that the maximum electroporation efficiency was achieved under the following conditions in a 0.2 cm electroporation cuvette containing CIK cells (1.5 x 10(7)/mL, 200 microl): electric voltage 200 V, pulse time 45 ms, plasmid 30 microg, and one electric shock. The total genomic RNA of grass carp reovirus (GCRV) was extracted in this experiment and reversely transcribed into cDNA, which was used to amplify the gene segment of GCRV non-structural protein NS26 using designed specific primers. The PCR product was recombined into pEGFP-N1 vector. The fusion protein EGFP-NS26 was successfully and efficiently expressed in the CIK cells by electroporation, which was confirmed by both fluorescent imaging and Western blot analysis. This experiment laid a foundation for further functional studies of the non-structural protein NS26 of GCRV.

Leptin activates STAT3 and ERK1/2 pathways and induces endometrial cancer cell proliferation.

Obesity is an established risk factor for endometrial cancer. Leptin, a secreted protein of the ob gene by white adipose tissue, plays an important role in the regulation of food intake and energy consumption in the brain and acts as a potential growth stimulator in normal and neoplastic cancer cells. However, a direct role for leptin in endometrial cancer has not been demonstrated. In the present study, the effect of leptin on the proliferation of Ishikawa endometrial cancer cells was investigated as well as the possible mechanism(s) underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors. The expression of leptin receptor (ObRb) in Ishikawa cells was detected by RT-PCR and Western blotting. The cells after serum starvation, were treated by leptin with various concentrations (0, 10, 50, 100, 150 ng/mL) for different durations (6, 12, 24 h). The effect of leptin treatment on cell proliferation was examined by MTT assay. Meanwhile, inhibitory effect of Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) inhibitor AG490 or extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 on the proliferation of Ishikawa cells induced by leptin was also studied. Ishikawa cells were treated with 100 ng/mL leptin for various periods (0, 20, 40, 60 min), and the levels of STAT3 phosphorylation and ERK1/2 phosphorylation were examined by Western blotting. The results showed that leptin induced the phosphorylation of STAT3 and the activation of ERK1/2 in a time- and dose-dependent manner in the Ishikawa endometrial cancer cells. Blocking STAT3 phosphorylation with the inhibitor AG490, or blocking ERK1/2 activation by the specific ERK1/2 kinase inhibitor, PD98059, abolished leptin-induced proliferation of Ishikawa cells. In addition, leptin was found to potently induce the invasion of endometrial cancer cells in a Matrigel invasion assay. Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of STAT3 (AG490) and ERK1/2 kinase (PD98059). These results suggested that leptin promotes endometrial cancer growth and invasiveness by activating STAT3 and ERK1/2 signaling pathways and therefore blocking its action at the receptor level can be a rational therapeutic strategy.

Leptin Activates STAT3 and ERK1/2 Pathways and Induces Endometrial Cancer Cell Proliferation / 华中科技大学学报（医学）（英德文版）

Obesity is an established risk factor for endometrial cancer.Leptin,a secreted protein of the ob gene by white adipose tissue,plays an important role in the regulation of food intake and energy consumption in the brain and acts as a potential growth stimulator in normal and neoplastic cancer cells.However,a direct role for leptin in endometrial cancer has not been demonstrated.In the present study,the effect of leptin on the proliferation of Ishikawa endometrial cancer cells was investigated as well as the possible mechanism(s) underlying this action in endometrial cancers which express both short and long isoforms of leptin receptors.The expression of leptin receptor (ObRb) in Ishikawa cells was detected by RT-PCR and Western blotting.The cells after serum starvation,were treated by leptin with various concentrations (0,10,50,100,150 ng/mL) for different durations (6,12,24 h).The effect of leptin treatment on cell proliferation was examined by MTT assay.Meanwhile,inhibitory effect of Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) inhibitor AG490 or extracellular signal-regulatedkinase 1/2 (ERK1/2) inhibitor PD98059 on the proliferation of Ishikawa cells induced by leptin was also studied.Ishikawa cells were treated with 100 ng/mL leptin for various periods (0,20,40,60 min),and the levels of STAT3 phosphorylation and ERK1/2 phosphorylation were examined by Western blotting.The results showed that leptin induced the phosphorylation of STAT3and the activation of ERK 1/2 in a time-and dose-dependent manner in the Ishikawa endometrial cancer cells.Blocking STAT3 phosphorylation with the inhibitor AG490,or blocking ERK1/2 activation by the specific ERK1/2 kinase inhibitor,PD98059,abolished leptin-induced proliferation of Ishikawa cells.In addition,leptin was found to potently induce the invasion of endometrial cancer cells in a Matrigel invasion assay.Leptin-stimulated invasion was effectively blocked by pharmacological inhibitors of STAT3 (AG490) and ERK1/2 kinase (PD98059).These results suggested that leptin promotes endometrial cancer growth and invasiveness by activating STAT3 and ERK1/2 signaling pathways and therefore blocking its action at the receptor level can be a rational therapeutic strategy.

Effect of hormone replacement therapy on serum complement (C3, C4) and immunoglobulin (IgG, IgM) levels in post-menopausal women.

Serum C3, C4, IgG and IgM levels were evaluated in healthy post-menopausal women receiving short-term hormone replacement therapy (HRT) regimens and in untreated women. Serum C3, C4, IgM and IgG levels were assessed in 54 women receiving HRT therapy (CEE 0.625 mg+MPA 2.5 mg/day), and in 54 control women not receiving HRT. The results showed that the mean serum C3 and C4 levels were significantly higher in women receiving HRT than those untreated women (P<0.01). There was significant difference in IgG and IgM levels between two groups. It was concluded that HRT might be involved in the development of cardiovascular diseases through inflammatory mechanisms, as suggested by increased serum levels of C3 and C4.

Effect of Hormone Replacement Therapy on Serum Complement (C3,C4) and Immunoglobulin (IgG, IgM) Levels in Post-menopausal Women / 华中科技大学学报（医学）（英德文版）

Serum C3, C4, IgG and IgM levels were evaluated in healthy post-menopausal women receiving short-term hormone replacement therapy (HRT) regimens and in untreated women. Serum C3, C4, IgM and IgG levels were assessed in 54 women receiving HRT therapy (CEE 0.625 mg+MPA 2.5 mg/day), and in 54 control women not receiving HRT. The results showed that the mean serum C3 and C4 levels were significantly higher in women receiving HRT than those untreated women (P＜0.01). There was significant difference in lgG and IgM levels between two groups. It was con-cluded that HRT might be involved in the development of cardiovascular diseases through inflamma- tory mechanisms, as suggested by increased serum levels of C3 and C4.

Expression of leptin long-form receptor mRNA in luteinized granulosa cells of obese women with polycystic ovary syndrome.

To investigate the expression of mRNA of leptin long-form receptor (OB-Rb) in luteinized granulosa cells of obese women with polycystic ovary syndrome (PCOS), and to determine the role of leptin in the physiopathology of PCOS, luteinized granulosa cells were collected from the follicle fluid of 10 obese women who met the diagnostic criteria for PCOS and their BMI was equal to or greater than 25 kg/m(2), and at the same time, granulosa cells were collected from 10 normal women undergoing IVF-ET who served as the control group. Some luteinized granulosa cells were taken from normal women for in-vitro culture, into which human leptin of different concentrations was added (0, 10, 100 and 1000 ng/mL). After stimulation with leptin for 48 h, RT-PCR was employed for the detection of the expression of OB-RLmRNA in the luteinized granulosa cells. Our results showed that the level of OB-RLmRNA in luteinized granulosa cells of obese PCOS women was higher than those in the control (P<0.05). In luteinized granulosa cells cultured in vitro and stimulated by human leptin for 48 h, the level of OB-RLmRNA was higher than those without leptin stimulation (P<0.01), and when leptin concentration was at 100 ng/mL, and the level of OB-R(L)mRNA reached a peak. It is concluded that in obese PCOS women, the level of serum leptin is increased, which promotes the expression of OB-R(L) in luteinized granulosa cells and increases the sensitivity of the granulosa cells to leptin. Leptin may contribute to anovulation in obese women with PCOS.

Effects of metformin combined with cyproterone acetate on clinical features, endocrine and metabolism of non-obese women with polycystic ovarian syndrome.

In order to explore the effects of metformin combined with cyproterone acetate (CPA) on the clinical features, endocrine and metabolism of the patients with polycystic ovarian syndrome (PCOS), 50 cases of non-obese PCOS were randomly subjected to CPA (CPA treatment group, n = 25) and CPA+ metformin (n = 25) treatment for 6 months. Before and after treatment the body mass index (BMI), waist : hip ratio (WHR), ovarian volume, serum gonadotrophin, androgen and sex hormone-binding globulin (SHBG) levels, and fasting lipid, glucose and insulin levels were measured. The results showed that all of the parameters in two groups were similar before treatment. After treatment for 6 months in the CPA+ metformin group, BMI and WHR were significantly decreased, while insulin sensitivity was significantly decreased as Compared with those before treatment. In CPA group, no significant changes were found before and after treatment. Combined use of CPA and metformin could result in the reduction of serum androstenedione and increases of serum SHBG levels as compared with the CPA treatment alone. It was concluded that combined use of CPA and metformin could improve the insulin sensitivity, and further suppress the hyperandrogenism in non-obese women with PCOS.

Mechanism of elevated vascular endothelial growth factor levels in peritoneal fluids from patients with endometriosis.

In order to investigate the mechanism of elevated vascular endothelial growth factor (VEGF) in peritoneal fluids from patients with endometriosis, macrophages were recovered from peritoneal fluids obtained at the time of diagnostic laparoscopy from infertile women with endometriosis (EMT group, n=20) and without endometriosis (control group, n=20). Macrophages were cultured in vitro. The VEGF levels of peritoneal fluid and the supernatant of macrophages culture were determined by enzyme linked immunoassay (ELISA). Meanwhile, the eutopic (n=20) and ectopic endometrium (n=20) from endometriosis patients, and normal edometrium (n=20) from non-endometriosis patients were obtained for the analysis of VEGF expression by labeled Streptavidin Biotin (LSAB). It was found that VEGF levels in peritoneal fluid and macrophages culture supernatant were significantly higher in EMT group than in control group (P<0.01). In normal endometrium, VEGF showed a cyclic changes and similar in eutopic and ectopic endometrium from patients with endometriosis. There was no difference in the intensity of VEGF in endometrium between two groups within each menstrual phase. It is suggested that altered VEGF production by peritoneal macrophages and ectopic endometrium secretion may contribute to the elevated VEGF levels in the peritoneal fluid of patients with endometriosis.