Methylation Of Promoter And Expression Of Steroidogenic Factor-1 In Cultured Granulosa Cells From Women With Endometriosis

Part I Effect of endometriosis on the clinical outcome of in-vitro fertilization and embryo transferAim:To compare the clinical outcome of in-vitro fertilization and embryo transfer(IVF-ET) in women with and without endometriosis.Methods: 107 infertile women with laparoscopically diagnosed endometriosis and 121 controls without endometriosis undergoing IVF-ET were included for the retrospective analysis.The primary outcomes were hormone profiles, duration of stimulation, total gonadotropin dose requirement, peak serum estradiol level, total number of oocytes retrieved, fertilization rate, embryo quality, implantation rate, and clinical pregnancy rate.Results: There were no differencesbetween the two groups with respect to age, infertility duration, body mass index(BMI), baseline serum FSH level, endometriumthickness and fertilization rate. However, women with endometriosis required significantly higher FSH doses(P<0.05) than controls despite that the number of oocytes obtained and the peak serum estradiollevel were comparable.The number of high-quality embryos(P<0.01) and frozen embryos(P<0.05) in the endometriosis group wassignificantly lower than that of the control group. Although a similar number of embryos was transferred in each group, implantation and clinical pregnancy rates showed a decreasing trend in the endometriosis group without reaching statistical significance.Conclusion: Endometriosis showed negative effect on IVF-ET outcome which presented the lower percentage of high-quality embryos and the number of frozen embryos, also the women with endometriosis had decreased ovarian response to gonadotropins compared with controls.Part II Steroidogenic factor-1 and liver receptor homologue-1expression in cultured granulosa cells from women with endometriosisAim: To investigate the expression of aromatase(via CYP19 and the CYP19 II promoter) and the orphan nuclear receptor family members, steroidogenic factor-1(SF-1) and liver receptor homologue-1(LRH-1)in cultured luteinized granulosa cells from women with endometriosis.Methods: Luteinized granulosa cells from patients undergoing IVF(16 patients with endometriosis and 28 controls) were examined for the m RNA expression of CYP19, CYP19 PII, SF-1 and LRH-1 which determined by quantitative reverse transcription-polymerase chain reaction(QT RT-PCR). Student’s t-test, the Mann-Whitney U test and Spearman’s rank correlation were used for statistical analysis.Results: The m RNA expression levels of CYP19, CYP19 PII, SF-1and LRH-1 in granulosa-lutein cells were decreased in women with endometriosis compared to the control group. The simultaneous down-regulation expression of SF-1, and LRH-1 CYP19 PII in endometrioticgranulosa cells indicated their positive correlation.Conclusion: The present results demonstrated the aberrant expressions of SF-1 and LRH-1 in endometriotic granulosa-lutein cells. This finding may be helpful in understanding infertility associated with endometriosis and reduced P450 aromatase activity in endometriotic granulosa cells.Part III Methylation of the 5’ Cp G sites of Steroidogenic factor-1 in endometriotic granulosa cellsAim: The objective of the study was to gain further insight into the mechanism for differential SF-1 expression in normal and endometriotic granulosa cells.Methods:We evaluated the methylation status of 5’ Cp G sites of SF-1 promoter in normal and endometriotic granulosa cells using Bisulfite Sequencing PCR.Results:Methylation status of a total of 13 Cp G sites across a 213-bp region in the approximately 252-bp Cp G sites(-84/+186) at the SF-1 promoter was characterized by bisulfite genomic sequencing. Ten clones were checked for each involved subject. The 13 Cp G sites of SF-1 were differentially methylated in normal group. However, increased methylation in endometriotic granulosa cells was observed when compared with normal granulosa cells(P<0.05). There found eleven significantly hypermethylated sites including Cp G+7,+18,+21,+42, +54,+60, +77,+121,+132,+141 and +146(P<0.05).Conclusion(s): We demonstrated that hypermethylation status of 5’ Cp G site of SF-1 promoter in endometriotic granulosa cells, which may explain the mechanism for reduced SF-1 m RNA expression in granulosa cells of endometriosis.