| Objective: To observe the effects of bushen and shugan on IVF-ET inpatients with pregnancy outcom.The relationship between improve thepregnancy outcome role to improve the quality of oocytes and its relationshipSmads signal pathway that two method, Compare the reduced mechanism ofsimilarities and differences,the clinical application of treatment of ovulationdisorder to provide experiment basis by Bushen treatment and Shugantreatment.Methods: Sixty infertile patients meet criteria for inclusion and exclusioncriteria from Bethune International Peace Hospital from February,2010toOctober,2011undergoing the treatment of IVF-ET, Sign the informed consentform all patients should be approved by the hospital ethics committee，femalepatients with bushen deficiency of TCM standards of20cases, female patientswith shugan depression of TCM standards of20cases, female patients withthe control group of TCM standards of20cases. Bushen treatment (BushenTiaojing Recipe+GnRHa/FSH/HCG) and Shugan treatment (XiaoYao san+GnRHa/FSH/hCG).and control group (GnRHa/FSH/hCG). The content ofhormones(E2、 LH、 P)on HCG day, number of oocytes and matureoocytes,fertilization number were observed. Follicular fluid in ovarian folliclewere kept with18mm in diameter,the granalosa cells were separated fromfolliaular fluid comtaining granular cell used by person lymphocyteseparation.Tgranalosa cell used by person lymphocyte separation in patiantswith IVF-ET fubal facter from Bethune international peace hospital ofobstetrics and gynecology reproductive medicine center and four institute forreproductive medicine center of shijiazhuang. With the1640mediumcontaining10%fetal bovine serum granulosa cells, and adherent cells after24 h, join the bushen menstrual function respectively and a high concentrationof xiaoyao powder for bushen pill (18%), The concentration (12%), Lowconcentration (6%) people drug-containing serum processing cell6h,12h,24h,48h. Bushen respectively high, medium and low dose group and Shuganhigh, medium and low dose group.0h for normal serum group, named asblank control. GDF-9, BMPR-II, Alk5, Smad2/3/4mRNA expression weredefected by the RT-PCR methed to detect edge of follicular granulosa cells;GDF-9, BMPR-II, Alk5, Smad2/3/4of the protein content were defected byimmunofluorescence methods advantage of follicular granulosa.GDF-9,BMPR-II, Alk5, Smad2/3/4protein expression by incell-western methodscultured granulosa cells.Results:1The outcome effects of bushen, shugan, in patients with IVF-ET1.1Serum E2, P, LH levels in each group on HCG dayCompared with control group, bushen group serum E2increased (all P<0.05), with statistical significance. Compared with shugan group, bushenserum E2group decreased but no statistical significance (P>0.05). Comparedwith control group, bushen, shugan group serum LH, and P has no statisticalsignificance (P>0.05).1.2Number of oocytes, number of fertilization, number of high qualityembryo in serumCompared with control group, bushen, shugan group number of oocytessignificantly increased, with statistical significance (P <0.05). Compared withshugan group number of fertilization, bushen group significantly increased butwithout statistical significance (P>0.05). Compared with control group,bushen, shugan group number of high quality embryo significantly increased,with statistical significance (P<0.05). Compared with the shugan group:bushen group number of high quality embryo slightly reduced but withoutstatistical significance (P>0.05).2Bushen,shugan, granulosa cells in patients with IVF-ET GDF-9, BMPR-II, ALK5, Smad2and Smad3and Smad4mRNA expression Compared with control group, bushen and shugan group advantage GDF-9mrna expression in follicular granulosa cells obviously increased, withstatistical significance (P<0.05). Advantages compared with shugan group,bushen group follicular granulosa cells GDF-9mrna expression increased,with statistical significance (P<0.05).Compared with control group, bushen and shugan groups granular cellBMPR-II mRNA expression has a increased trend, but there is no statisticalsignificance (P>0.05).Compared with control group, bushen and shugan groups granular cellALK5mRNA expression has a increased trend, but there is no statisticalsignificance (P>0.05).Compared with control group, bushen and shugan group advantage offollicular granulosa cells Smad2mRNA expression obviously increased, withstatistical significance (P<0.05). Compared with shugan group, bushen groupadvantage follicular granulosa cells Smad2mRNA expression increased, withstatistical significance (P<0.05).Compared with control group, bushen and shugan group advantage offollicular granulosa cells Smad3mRNA expression obviously increased, withstatistical significance (P<0.05). Compared with shugan group, bushen groupadvantage follicular granulosa cells Smad3mRNA expression increased, withstatistical significance (P<0.05)Compared with control group, bushen and shugan group advantage offollicular granulosa cells Smad4mRNA expression has a increased trend, butthere is no statistical significance (P>0.05).3Bushen, shugan, granulosa cells in patients with IVF-ET GDF-9, BMPR-II, ALK5, Smad2and Smad3and Smad4protein expressionCompared with control group, bushen and shugan group advantage offollicular granulosa cells GDF-9protein expression increased, with statisticalsignificance (P<0.05). Compared with the shugan group, GDF-9proteinexpression increased bushen group, with statistical significance (P<0.05). Compared with control group, bushen and shugan group BMPR-2protein expression has increased trend, but there is no statistical significance(P>0.05). Each ALK5protein expression did not see the granule cells.Compared with control group, bushen and shugan group advantage offollicular granulosa cells Smad2and Smad3protein expression increased, withstatistical significance (P<0.05). Compared with the shugan group, bushengroup advantage follicular granulosa cells Smad2increased the proteinexpression was statistically significant (P<0.05).Compared with control group, bushen and shugan group advantage offollicular granulosa cells Smad4protein expression has increased trend, butthere is no statistical significance (P>0.05).4Bushen menstrual function, ease pill containing medicine serum on in vitrohuman ovarian granulosa cells GDF-9different time points, BMPR-II,ALK5, Smad2and Smad3and Smad4protein expressionCompared with0h, bushen in high, medium and low dose, shugan ofhigh, middle, low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48h GDF-9protein expression were increased, with statisticalsignificance (P<0.05).6h GDF-9protein expression was obviously higherthan12h,24h,48h, were statistically significant (P<0.05). And bushen inhigh, medium and low dose of Chinese medicine incubation granulosa cell6h,shugan of high, medium and low doses of GDF-9protein expressiondecreased, had statistical significance (P<0.05). And6h high dose of Chinesemedicine incubation bushen granulosa cells, bushen in middle and low dose ofGDF-9protein expression decreased, had statistical significance (P<0.05).High dose of Chinese medicine shugan and6h incubation granulosa cells,shugan medium and low doses of GDF-9protein expression decreased, hadstatistical significance (P<0.05).Compared with0h, bushen in high, medium and low dose, shugan ofhigh, medium and low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48h BMPR-II trends to increase protein expression has nostatistical significance (P<0.05). Compared with bushen in high, medium and low dose, shugan high, medium and low dose BMPR-2protein expressionhas increased trend, no statistical significance (P>0.05).Compared with0h, bushen in high, medium and low dose, shugan ofhigh, medium and low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48h ALK5protein expression decreased, with no statisticalsignificance (P>0.05). Compared with bushen in high, medium and low dose,shugan high, medium and low dose ALK5protein expression decreased, withno statistical significance (P>0.05).Compared with0h, bushen in high, medium and low dose, shugan ofhigh, medium and low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48h ALK5protein expression decreased, with no statisticalsignificance (P>0.05). Compared with bushen in high, medium and low dose,shugan high, medium and low dose ALK5protein expression decreased, withno statistical significance (P>0.05).Compared with0h, bushen in high, medium and low dose, shugan ofhigh, middle, low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48hsmad2protein expression were increased, with statisticalsignificance (P<0.05).48h Smad2protein expression was obviously higherthan6h,12h,24h, and were statistically significant (P<0.05). And bushen inhigh, medium and low dose of Chinese medicine48h incubation granulosacells, shugan of high, medium and low doses of Smad2protein expressiondecreased, had statistical significance (P<0.05). With high dose of Chinesemedicine incubation for48h bushen granulosa cells, bushen Smad2proteinexpression decreased, middle and low dose had statistical significance(P<0.05).48h with high dose of Chinese medicine shugan granular cell,incubation, low-dose Smad2protein expression in shugan decreased, hadstatistical significance (P<0.05).Compared with0h, bushen in high, medium and low dose, shugan ofhigh, middle, low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48h Smad3protein expression were increased, with statisticalsignificance (P<0.05).48h Smad3protein expression was obviously higher than6h,12h,24h, and were statistically significant (P<0.05). And bushen inhigh, medium and low dose of Chinese medicine48h incubation granulosacells, shugan Smad3high, medium and low doses of protein expressiondecreased, had statistical significance (P<0.05). With high dose of Chinesemedicine incubation for48h bushen granulosa cells, bushen Smad3proteinexpression decreased, middle and low dose had statistical significance(P<0.05).48h with high dose of Chinese medicine shugan granular cell,incubation, low-dose Smad3protein expression in shugan decreased, hadstatistical significance (P<0.05).Compared with0h, bushen in high, medium and low dose, shugan ofhigh, medium and low dose of Chinese medicine incubation granulosa cell6h,12h,24h,48h Smad4protein expression decline, no statistical significance(P>0.05). Compared with bushen in high, medium and low dose, shugan high,medium and low doses of Smad4protein expression, no statistical significance(P>0.05).Conclusions:(1)Bushen menstrual function, xiaoyao powder can improve IVF-ET inpatients with pregnancy outcomes, and improve the fertilization rate, increasethe optimal number of embryos.(2) Bushen menstrual function, ease pill roleto improve the quality of oocytes and raised GDF-9in granulosa cells andtheir Smads pathways of Smad2and Smad3mRNA and protein expression ofrelated.(3) Bushen menstrual function, ease pill in granulosa cells incubatedwith6hours when the GDF-9protein expression of peak and differentexpression in bushen and shugan group, the overall trend for bushen in highdose group is superior to shugan high, medium and low dose group.(4) Bushenmenstrual function, ease pill in granulosa cells incubated with48hours ofSmad2and Smad3protein expression of peak and different expression inbushen and shugan group, the overall trend for bushen in high dose group issuperior to shugan high, medium and low dose group.(5) Bushen menstrualfunction, ease pill may be by adjusting the oocyte GDF-9secretory factorsand signaling pathways Smad2and Smad3protein expression in order to improve control over stimulate ovulation IVF-ET in patients with the qualityof oocytes, and bushen in high dose is better than that in medium and low dose(6) GDF-9in granulosa cells and bushen group Smads pathway Smad2andSmad3mRNA and protein expression is higher than shugan group, speculatedthat the role of the bushen and regulate the menstrual function will improvethe quality of oocytes is better than the ease pill. |
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