| [Background]Androgens play important roles in follicular growth, development, and atresia, as well as in steroidogenesis. First, androgens act as essential substrates for the production of estrogen. Second, results of some animal experiments showed that Androgens appear to be positive regulators of follicular development by increasing the expression of FSHR and promoting the secretion of the IGF-â… in the granulose cells. IGF-â… showed some synergistic effects with FSH on follicuogeneisis. Therefore androgens may enhance follicular responsiveness and then promote the development of the follicles. At the same time, animal research also suggested that the androgen might have negative effect on the follicle development. In recent years, it has been reported that some clinical trials used the androgen priming protocol before or during the controlled ovarian hyperstimulation (COH) in IVF patients in order to increase the ovary response to the exogenous gonadotrophins. However, the outcomes of these clinical studies did not accord with each other. Our study was designed to explore the feasibility of androgen supplement before or during the IVF cycle to impove the IVF outcome.[Objective]1 To observe the serum total testosterone level during the controlled ovarian stimulation. To assess possible associations between total testosterone level and IVF stimulation parameters or IVF pregnancy outcome.2 To evaluate the relationship between the serum total testosterone level and the expression of the FSHR in the granulosa cells.3 To evaluate the effects of testosterone treatment for different doses and different incubating time on FSHR, IGF-â… and IGF-â…¡expression in human luteinized granulusa cells. [Methods]Our study was devided into three parts: Part one:A clinical prospective study was involved in this part. The serum total testosterone levels was measured during the IVF cycle. The quantity and the size of the follicles, the levels of sex hormones, and the amount of oocyte retrieved, mature oocyte, and blastomeres were recorded. Also the duration of controlled ovarian stimulation and the total rFSH doses were noted. The patients were devided into two groups according to the IVF outcome or the basal total testosterone level respectively. We described the changes of total testosterone level during the IVF cycle. Linear regression analysis was used to assess the correlation between linear data.Part two:Hormone levels and the ovarian response variables were recorded as in part one. In addition, preovulatory granulosa cells were collected from follilcular fluid obtained from patients undergoing transvaginal oocyte retrieval with untrasound guidance. According to the quantity of mature oocyte, the patients were grouped as normal response group, low response group and high response group. The mRNA level of FSHR in the granulosa cells from each patient was quantitatively measured using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). The clinical parameters and the expression of FSHRmRNA among the three groups were compared. And finally, linear regression analysis assessed the correlation between linear data.Part three:Preovulatory granulosa cells were collected from follilcular fluid obtained from patients undergoing transvaginal oocyte retrieval with untrasound guidance. After that, granulosa cells were cultured for 2 days in DMEM/F12 medium supplemented with 10%FBS, penicillin/streptomycin, and glutamine. The cells were grouped and incubated in DMEM/F12 medium supplemented with 1%FBS and different concentrations of testosterone (10-8 mol/L,10-7 mol/L,10-5mol/L) for another 6 or 24 hours. The mRNA levels of FSHR, IGF-I and IGF-II were were quantitatively measured using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We analyzed the effects of testosterone treatment with different doses and different cultured time on FSHR, IGF-I and IGF-II expression in granulusa cells.[Results]Part one:1 During the controlled ovarian stimulation (COH), total testosterone (TT) levels rised in accordance with the follicle development.2 The change of TT level during the COH presented a parallel phenomenon with the basal TT level.3 Patients were grouped by basal TT levels at increments of 0.1ng/ml from 0.2ng/ml to 0.4ng/ml. The basal TT levels did not significantly correlate with any of the stimulation parameters tested and the IVF outcome for each increment.4 TT levels during the COH correlated positively with the antral follicle count, the quantity of oocyte retrieved and mature oocyte. And it correlated negatively with the basal FSH level and the number of gonadrotropin ampoules used.5 TT level measured 14 days after oocyte retrivel in pregnancy group was significantly higher than that in the non-pregnancy group.Part two:1 The expression of FSHR was different among the three different ovarian response groups. FSHRmRNA level s in high response group was significantly higher than that in the low response group.2 The FSHRmRNA correlated negatively with the total doses of the rFSH. And it correlated positively with the peak E2 level on the day of hCG injection and oocyte number retrieved.3 The basal TT levels did not significantly correlate with both any of the stimulation parameters tested and the expression of FSHRmRNA. TT levels during the COH correlated negatively with the duration of COH and the number of gonadrotropin ampoules used. And it correlated positively with the peak E2 on the day of hCG injection, the quantity of oocyte retrieved, and the expression of FSHRmRNA.Part three:1 After the cells were incubated with testosterone of different concentrations for 6 hours, the expression of FSHRmRNA was significantly increased in a dose-depended way. However, when cultured for 24 hours, no statistical significant difference was observed among the three groups.2 After the cells were incubated with testosterone of different concentrations for 6 hours, no statistically significant difference were observed on the expression of IGF-ImRNA among the three groups. However, when cultured for 24 hours, the expression of IGF-ImRNA was significantly decreased in a dose-depended way.3 After the cells were incubated with testosterone of different concentrations for 6 hours, there were no statistically significant differences on the expression of IGF-IImRNA among the three groups. However, when cultured for 24 hours, the expression of IGF-IImRNA in 10-7 mol/L group was higher than that of the 10-8 mol/L group and 10-5mol/L group.[Conclusions]1:Basal serum total testosterone level may not predict the IVF outcome and the ovarian response.2:Total testosterone levels during the controlled ovarian hyperstimulation procedure correlated positively with the ovarian response. The expression of FSHRmRNA in human luteized granulosa cells correlated positively with the ovarian response.3:Testosterone of high concentration for short time could up-regulate the expression of FSHR and IGF-II. But when treated for long time, the up-regulation of the expression disappeared. Testosterone of high concentration could down-regulate the expression of IGF-I, and the down-regulation became stronger as treated for a longer time.[Key words]IVF, testosterone, ovarian response, granulosa cell, FSHRmRNA,IGF-â… mRNA,IGF-â…¡mRNA...
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