Abstract
摘要:近年冷凍卵子在國內外已蔚為風潮,不但成為女性臨界卵子老化年齡卻苦無適當生育機會時具有彈性調整生涯規劃的另一種選擇,同時也為癌症病患在面對治療而必須摘除卵巢時,提供保留生育能力的契機。一般在不孕症中心進行不孕症治療的取卵過程中,約有15~30%為無法使用的未成熟卵。此等卵子提供珍貴的臨床與研究材料來源。儘管越來越多科學家與臨床醫師對此等卵子表示高度興趣,然而,未成熟的冷凍卵子在體外成熟後的效果,卻令人大失所望,導致其在真正臨床應用上大多遭到棄置的命運。再則,文獻上亦普遍缺乏對此等未成熟卵子於體外成熟與玻璃化冷凍後之卵品質與功能評估與改善的基礎研究。
卵子成熟為一短暫卻嚴謹的生物調節過程,為後續其能逐漸獲取發育能力之關鍵。玻璃化冷凍定義為最大程度地提高冷凍液體黏稠度,使其在低溫狀態下固化而無冰晶之形成,進而可暫時終止任ㄧ進展中的生物過程。因此,冷凍解凍過程對於卵母細胞體外成熟作用將造成至少兩倍以上的破壞,包括:1). 由於細胞環境處於極端劇烈的生理變化(溫度,滲透壓等),所導致細胞組成(如:細胞骨架)的破壞;2). 干擾後續成熟作用之進展。故未臻理想之體外成熟系統加上冷凍解凍破壞之雙重因子作用下,降低其受精與後續胚胎之發育潛能,其牽涉之機制如紡垂體結構及其生合成過程的破壞,染色體不對稱分離,產生非整倍染色體之異常胚增加,最後導致胚在子宮的著床率低下。
我們的長期目標為發展出理想的體外成熟系統配合玻璃化冷凍解凍程序提供有效的保存珍貴的卵子。在此計畫目標首先有系統地於細胞層次證明一般成熟卵子與未成熟卵子在冷凍解凍過程中的破壞情形,進而探討玻璃化冷凍時機及其對體外成熟後續發育結果的影響。計畫中將針對紡錘體與DNA的完整性,及其於單一精子注射後,此等卵子的胚體外發育及其胚移植後續產生子代的能力。透過本計畫之執行, 若此等核心技術在小鼠模式中能順利被建立, 除了針對體外成熟過程遭遇的冷凍傷害 於細胞層次首次系統性地得到證明,而計畫最終目標所探討之冷凍未成熟卵子與體外成熟後的卵品質與功能,亦透過子代的產生更得到進一步的實質驗證。更重要的,透過本計畫之執行,將提供未來在人類不孕症治療臨床應用相關技術之改進外,並於建立人類“卵子銀行“時的卵品質安全與功能評估提供關鍵訊息。
Abstract: Oocyte vitrification has become a reality and is used as an adjunct to human in vitro fertilization (IVF). In Taiwan and worldwide, banking eggs is a lifestyle choice for many women especially those who want to delay childbirth yet without “jumping” the fertility cliff when they are 35 years or older. It is also an invaluable option for cancer patients who have to undergo oophorectomy (i.e. removing an ovary or ovaries).
In a routine IVF clinical cycle, 15-30% oocytes are immature (i.e. MI oocytes) and not used. They represent precious clinical and research resources. In vitro maturation (IVM) of vitrify-thawed MI oocytes, however, has been a challenge despite increasing interests from clinics and researchers to use these otherwise discarded materials. Indeed, there is a lack of basic research on evaluating and improving the quality and function of immature oocytes after IVM and vitrification.
Oocyte maturation is a temporally strictly regulated process that is crucial for the gradual acquisition of developmental competence. Vitrification, defined as the solidification of a solution at low temperature by extreme elevation in viscosity during cooling without ice crystallization, suspends any biological process. Therefore vitrification/thawing in the context of IVM may render at least two folds of damages to the oocytes: (1) causing structural damages to the cellular components such as cytoskeleton, due to the drastic physical changes of the cellular environment; (2) interrupting the otherwise continuous maturation process. Together, un-optimized interplay of IVM and vitrification/thawing may compromise the fertilization and subsequent embryo development capacity of these oocytes, through mechanisms such as disturbed spindles dynamics and structures, and chromosome miss-segregation, leading to increasing risks of aneuploidies in the embryos, and ultimately low implantation rates.
Our long-term goal is to develop a working protocol of IVM on vitrify-thawed MI oocytes. In the present work, we will first work to document the damages of vitrification/thawing on matured and immature oocytes. Next, we will determine the effects of timing of vitrification on the outcome of IVM. The parameters that we will investigate include spindles and DNA integrity, embryo development rates, and term rates after intracytoplasmic sperm injection (ICSI) and embryo transfer.
Success of the proposed work will not only for the first time systemically document cryo-damages to the IVM process at a cellular level but also provide proof of principle by the generation of live-offspring from the vitrify-thawed IVM oocytes. Importantly, knowledge gained from this project will shed light on our understanding on vitrification and IVM of human oocytes and provide clues to improve these procedures in humans.
Keyword(s)
卵子
玻璃化冷凍
體外成熟
單一精子注射
胚發育
Oocyte
vitrification
in vitro maturation (IVM)
intracytoplasmic sperm injection (ICSI)
embryo development