| Part 1 The relationship between the expression of cytokines in mural granulosa cells and ovarian respondence and the pregnancy rate in IVFOBJECTIVETo investigate the expression of IL-1α,IL-1β,IL-1ra,IL-6,IL-17,VEGF,EGF,leptin and bFGF expressed in mural granulosa cells. Investigate the relationship between the expression of cytokines in mural granulosa cells and ovary respondence and the pregnancy rate in IVF.MATERIALS AND METHODS1. Mural granulosa cells examples: Human mural granulosa cclls (MGC) were rcceived from follicular fluid of women accepted IVF-ET in NanFang hospital from 2006.3 to 2006.9. The thirty-two cases of MGCs from ovulatory women with infertility causcd by male factor or tubal factor.2. MGCs culture in vitro: Granulosa cells were obtained from follicular fluid aspirated during oocyte retrieval, and serum E2 was detected just at thc time of oocytc retrieval. According to the numbcr of follicles≥14mm obtained during oocytc retrieval, the patients were divided into three groups: high responders (number=14), normal responders (number 5-13) and poor rcspondcrs (number=4). 35 days after embryo transfer, ultrasound-checked fetal heart throb definited pregnancy, contrary to that as the non-pregnant group. Collect the supernatant for detection of the value of cytokines.3. Luminex xMAP technology was used to measure the concentrations of IL-1a, IL-1β, IL-1ra, IL-6, IL-17, VEGF, EGF, bFGF, leptin in MGC samples simultaneously, then compared their variety in different groups.STATISTICAL METHODData were evaluated using SPSS 10.0 software. Independent-samples T test was used to compare concentrstions of cytokines in different age groups and pregnancy or not. One-way ANOVA were performed to analyse their variety in different oocyte respondence groups. Spearman's correlation was used to analyse relationship of cytokines. P≤0.05 was accepted as indicative of stastical significance.RESULTS1. IL-6,VEGF,leptin and bFGF were diversity significant between the women≤35 group and≥36 group (p＜0.05).2. IL-1a,IL-6 and VEGF were notable correlated with the number of eggs and E2 (p＜0.05). IL-1βand bFGF were significant correlated with the number of eggs (p＜0.05).3. Among different ovarian responders, the expression of IL-1a,IL-1β,IL-1ra,leptin,bFGF and VEGF was significantly variance (p＜0.05).4. In normal ovarian responsc group, IL-1a and IL-6,VEGF, IL-1βand IL-1ra,IL-6, IL-6 and IL-1ra, IL-6 and VEGF correlated significantly. The significantly negative correlation between leptin and IL-6 were closely related (p＜0.05).5. The value of IL-1a,IL-1β,IL-6,VEGF from the women who succeeded in becoming pregnantcy was higher than those who failed, but EGF and IL-1ra was oppositely (p＜0.05). CONCLUSIONS1. With increasing age, MGC of elderly patients secretion of IL-1 beta, IL-6, leptin and bFGF decreased.2. IL-1α, IL-1β, IL-1ra, IL-6, VEGF, Leptin and bFGF was significant difference in different ovarian respondence groups, suggesting that these cytokines related with ovarian responsiveness. MGC from the high responders secreted more cytokines.3. Through IVF-ET was treated pregnancy than non-pregnant women who MGC higher secretion of IL-1 Europium. IL-1 beta, IL-6 and VEGF and IL-1ra and EGF contrary. Suggest IL-1 Europium, IL-1β, IL-6 and VEGF, to oocyte development and the future growth and development of the embryo.4. Luminex liquid protein microarray analysis system has good flexibility, throughput, high sensitivity, Characteristics of a good signal-to-noise ratio, it could also detect the same number of samples of a very small amount of protein molecules, Other than the detection method has a number of advantages.Part 2 The relationship between the expression of cytokines in cumulus cells and ovarian respondence and the pregnancy rate in IVFOBJECTIVEStudy the expression of IL-la,IL-1β,IL-1ra,IL-6,IL-17,VEGF,EGF,leptin,bFGF expressed in cumulus cells. Investigate the relationship between the cytokines and ovary respondence and the pregnancy rate in IVF.MATERIALS AND METHODS1. Cumulus cells samples: Cumulus cells wrer taken from the follicular fluid of women through IVF treatment in the Reproductive Center of the south hospital. A total of 13 cases, both male or tubal factor infertility factors were. 2. Method of cultured cumulus cells: mechanically separated of the cumulus cells corporations around the egg, 0.1% hyaluronidase digestion and wind and percussion with suction repeatedly until cumulus cell particles dispersed as a single cell. By living cells density of 1×104/mL were seeded in 24-well culture plates, plus a bottle 1ml cell suspension. Joined with 10% fetal calf serum in 1ml RPMI 1640, 95% air, 37℃, saturation humidity conditions under cultivation after the adherent cells in serum-free RPMI-1640 replacement, 24 hours after the collection of the supernatant.3. Luminex xMAP technology was used to measure the concentrations of cytokines.STATISTICAL METHODData were evaluated using SPSS 10.0 software. Independent-samples T test was used to compare concentrstions of cytokines in pregnancy or not. One-way ANOVA were performed to analyse their variety in different oocyte respondence groups. Spearman's correlation was used to analyse relationship of cytokines. P≤0.05 was accepted as indicative of stastical significance.RESULTS1.IL-1ra secreted by CC was correlated significantly with mature eggs (p＜0.05). VEGF related closely with the number of mature eggs and estradiol (p＜0.05).2. According to the different number of follicle≥14mm, divided paients into 3 groups: low-responsed (number=4), normal-responded (number from 5 to 13) and the high-responsed (number=14). Three group patients with no significant difference between age (p＞0.05). VEGF in the groups are significantly different (p＜0.05).3. Age, the number of oocytes retrieved, estradiol and progesterone were no significant differences between pregnant and non-pregnant women (p＞0.05). Two cumulus cell culture supernatant of cytokines were similar level (p＞0.05 ). CONCLUSIONS1. IL-1ra secretion level of cultured human cumulus cell was relative to the number of mature eggs. VEGF relevanted with number of eggs,mature eggs and estradiol. IL-1ra and VEGF may controlled oocytes maturity.2. Age was similar in the different ovarian reactive groups. The expression of VEGF of cumulus cells was significant difference, but other cytokines were't, suggesting that VEGF played an important role in ovarian reaction.3. These nine factors secreted by cumulus cells were no significant relationship with clinical pregnancy rate, prompted expression of the above nine factors from cumulus cells did not affect IVF-ET in the clinical pregnancy rate.Part 3 Regulation of gonadotropin-releasing hormone agonist on the cytokines secreated by mural granulosa cellsOBJECTIVEStudy the regulation of gonadotropin-releasing hormone agonist to multiple cytokines and growth factors from mural granulose cells, for explored whether GnRHa application in IVF-ET safe or not.MATERIALS AND METHODS1. The method of collected mural granulose cells was same to part 1.2. Culturedgranulosa cells in vitro: after mural granulosa cells adhered culture capsule, GnRHa (Gonadotrophinreleasing hormone agonist, GnRHa) group was replaced serum-free RPMI-1640 added with GnRHa, the final concentration of rations100pg/ml, but the control group was't. After 24 hours cultured, the supernatants were collected.3. Lumunex system detected samples. STATISTICAL METHODData were evaluated using SPSS 10.0 software. T test was used to compare concentrstions of cytokines in the GnRHa group and the control group. P≤0.05 was accepted as indicative of stastical significance.RESULTSCompared cytokines secretion from GnRHa group and control group, eggs, mature eggs and pregnancy were no significant difference(P≤0.05), and cytokines secretion were no difference too(P≤0.05).CONCLUSIONSGnRHa had no significant impact on MGC expression of IL-1a, IL-1β, IL-1ra, IL-6, IL-17, EGF, and bFGF leptin, suggested in the IVF treatment application of GnRHa on ovarian cytokine secretion of granulosa cells and in control oocytes hadn't adverse effects. |
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