- 學位論文
體外培養液中的氧化自由基對於配子與 胚胎的作用
The effect of reactive oxygen species on gametes and embryos within in vitro culture media
摘要
研究目的:氧化自由基(Reactive oxygen species)(ROS)在精子活化和染色質的損壞扮有關鍵的角色，加入抗氧化物和血清合成添加物以保護精蟲的染色質和活化免於破壞。因此設計本研究計畫旨在釐清胚胎培養液微環境中氧化自由基水平及及精子準備培養液中加入抗氧化物或血清替代補充物如(Serum substitute supplement) (SSS)及血清蛋白替代物(Serum protein substitute)(SPS)對於精蟲活化和胚胎發育的影響。 研究方法及資料:在159(78/81)[78體外受孕組(In vitro fertilization) ( IVF) ; 81精子顯微注射組[(In cytoplasmic sperm injection)(ICSI)]接受體外輔助生殖科技治療週期不孕症病患在兩種準備胚胎培養液QAC/QAB(Q系列胚胎早期發育及囊胚分裂期培養液)和G1.3/G2.3 (G系列早期胚胎發育及囊胚分裂期培養液)，比較這兩種培養液中氧化自由基對胚胎發育有何影響有。另外收集30位不明原因不孕男性病患在精液準備培養液中加入抗氧化物或血清替代補充物及血清蛋白替代物後分析其氧化自由基水平對於精子活化作用和受孕能力。 研究結果:在體外受孕組和精子顯微注射組都呈現較多好的胚囊:在IVF cycle (26.2 ± 10.8%，P =0 .023)，在ICSI cycle : (33.3 ± 11.3%，P= 0 .033)。以評估氧化壓力加入精液分析項目的必要性。除此之外在不明原因不孕配偶其精液分析正常男性，在體外精子準備培養液中加入合成血清添加物(分為4組:HTS，HTP，QFS，QFP)及相對的抗氧化物(谷胱胺肽)，其中我們可發現在人類輸卵管液加血清替代補充物HTF+SSS(HTS)組其氧化自由基的量比QF+SPS (QFS)組較高(11,725 ± 1,172 vs. 6,278 ± 864 RLU)(relative light units)。其對精蟲活化包括精子活動力增加。同時色胺酸(Tyrosine)磷酸化程度、頂體反應及精子穿透卵子的能力也都較好。另外在HTS組和GSH組(HTS入抗氧化劑如谷胱胺肽)的比較，其結果亦是如此。 結論和建議:上述研究都和氧化自由基的高低程度有關，所以評估氧化壓力如氧化自由基的測試加入精液分析項目的必要性才不致影響精子活化測試的結果，是可加以考慮並可提供不明原因不孕症男性病患在接受何種輔助生殖科技(ART)治療方法才是最為恰當的一重要指標。
並列摘要
Object : Reactive oxygen species (ROS) is a key player for chromatin damage and capacitation of spermatozoa。The supplement of antioxidants and synthetic serum supplements during sperm preparation may benefit infertile couples。In this proposal，we will evaluate the effect of ROS capacity on sperm capacitation in order to determinate the necessity to measure ROS levels in semen analysis and the effect of embryo development。 Methods and Materials : We collected 159[(78 (In vitro fertility)(IVF)/ 81 (In cytoplasmic sperm injection)(ICSI)]treatment cycles。First，the level of ROS of two medias were checked then the retrieved ovum were set in the prepared QAC/QAB (Quinn's Advantage Cleavage and Blastocyst medium)media and the G1.3/G2.3 (GIII series medium for cleavage and blastocyst stage) media。After fertilization，We observed the development condition of embryo and compared the difference。Furthermore，we recruited 30 unexplained infertility male patients with normal results of semen analysis to evaluate the effect of ROS level and antioxidant (GSH，synthetic substitute supplement ; serum protein substitute ) in four sperm prepared culture medias(HTS，HTP，QFS and QFP)。 Result : We found QAC/QAB (Quinn's Advantage Cleavage and Blastocyst medium) media contain lower amounts of ROS than the G1.3/G2.3 (GIII series medium for cleavage and blastocyst stage) media。In IVF: Higher good blastocyst rates in IVF cycle: (26.2 ± 10.8%，P =0 .023)，In ICSI cycle: (33.3 ± 11.3%，P =0 .033) was the same when using QAC/QAB media than using G1.3/G2.3 media。Then，We also noted HTF+SSS(human tubal fluid plus serum plus serum substitute supplement)group had significantly higher ROS levels than QF+ SPS (Quinn's Advantage™ Fertilization plus serum protein substitute)group did (11,725 ± 1,172 vs. 6,278 ± 864 relative light units)。In addition，the spermatozoa cultured in QF+SPS had lower motility，Acrosome reaction rates and tyrosine phosphorylation levels compared with those cultured in HTS + SSS。The results as same between the HTF group and GSH group(HTF with glutathione)。 Conclusion and suggestion: These differences may lead to variations in spermatozoon ROS levels，thus affecting sperm function test results。It may offer an important marker to select what kind of therapeutic method for male partner of UI(Unexplained infertility) patient is the best and suitable。