- 學位論文
年齡與破卵針療程對人類卵丘細胞全甲基化與濾泡相關基因表現之相關性探討
Associations between follicle-related genes expression and DNA global methylation with age and oocyte retrieval in the human cumulus cells
摘要
作為治療不孕症之一的試管嬰兒療程會利用藥物或針劑給予賀爾蒙，對女性患者的次級卵母細胞及包覆在外圍給予支持和養分的卵丘細胞（Cumulus cell）刺激，若刺激過量則可能使患者產生卵巢過度刺激症候群（Ovarian Hyperstimulation Syndrome,OHSS）。其中試管嬰兒療程最後的破卵針即是因早期僅使用hCG造成患者產生OHSS併發症機率提高，現今多改合併使用GnRH agonist藉以降低OHSS發生風險。另一方面DNA甲基化（DNA methylation）是能穩定調控基因活性進而影響生物表現的重要機轉，在接受療程刺激後是否造成全甲基化程度的改變進而對卵丘細胞內的基因表現造成影響是為本研究探討的目標。 本碩論與中山醫學大學附設醫院生殖醫學中心合作，自2014年11月25日至2020年10月19日間共收集108個不同荷爾蒙治療的試管嬰兒療程個案，透過萃取其中的DNA及RNA並利用5-mC DNA ELISA定量細胞內DNA甲基化的程度，並利用Q-PCR（Real-time Quantitative Polymerase Chain Reaction）觀測11個濾泡相關基因—CYP11A1、CYP19A1、VEGF、HSD3B1、STAR、inhibinβ、LHCGR、FSHR、AREG、EREG及EGFR的表現。與108個個案之臨床資料結合分析，藉以在接受人工生殖療程的人類卵丘細胞中探討濾泡相關基因表現以及DNA甲基化之影響。 在全甲基化程度分析中發現，破卵針療程中合併使用GnRH agonist的族群全甲基化程度顯著提高，同時除了與LH直接相關的LHCGR外，其餘10個基因表現皆顯著較僅使用hCG的族群低。而在年齡的基因表現分組分析中不但觀察到FSHR及HSD3B1的表現降低與年齡上升呈現相關性，EREG更在35歲以上屬高齡孕婦的族群中有顯著表現較低的現象。綜合以上推論，年齡或是療程刺激的改變都有可能對卵丘細胞內全甲基化與基因表現造成影響，然而全甲基化與基因表現直接的關聯性與對於卵丘細胞的影響還待後續進一步的實驗證實。
並列摘要
As one of the treatments for infertility, the IVF course uses drugs or injections to give hormones to stimulate the secondary oocytes and the cumulus cells that are coated on the periphery to give support and nutrients. Excessive stimulation may cause patients to produce Ovarian Hyperstimulation Syndrome（OHSS）. Retrieval egg, the last step of IVF, in the early use of hCG only causes the patient to have an increased risk of OHSS complications. Nowadays, more changes are combined with the use of GnRH agonist to reduce the risk of OHSS. On the other hand, DNA methylation is an important mechanism that can stably regulate gene activity and affect biological performance. This research want to know the changes in the degree of global methylation after receiving a course of stimulation, will affect the gene expression in cumulus cells or not. Therefore, we collaborated with the Center for Reproductive Medicine of Chung Shan Medical University Hospital from November 25, 2014 to October 19, 2020 and we have collected 108 cumulus cells from different hormone treated IVF procedures and quantify the level of DNA methylation in the cells by using 5-mC DNA ELISA. At the same time, we used Q-PCR (Real-time Quantitative Polymerase Chain Reaction) to observe the performance of 11 OHSS-related genes- CYP11A1, CYP19A1, VEGF, HSD3B1, STAR, inhibinβ, LHCGR, FSHR, AREG, EREG and EGFR., and the data were combined with clinical data analysis to know how the OHSS-related genes expression and the DNA methylation in the human cumulus cells undergoing in vitro fertilization. In the analysis of the degree of global methylation, it was found that the degree of global methylation of the group combined with GnRH agonist during the treatment of the retrieval was significantly increased. At the same time, except for the LHCGR, which is directly related to LH, the gene expression of the other 10 genes was significantly lower than that of only hCG. In the group analysis of age, not only the decreased of FSHR and HSD3B1 gene expression was observed to be correlated with the increase in age, but EREG also had a significantly lower expression in the group of advanced maternal age. Based on the above result, changes in age or treatment course stimulation may have an impact on global methylation and gene expression in cumulus cells. However, the direct correlation between global methylation and gene expression and its impact on cumulus cells remains to be future work.