微環境自由基對試管嬰兒療程胚胎發育的作用

The effect of microenvironmental free radicals on embryo development for assisted reproduction technology cycles

李宗賢(Tsung-Hsien Lee)

指導教授 ： 楊友仕 ； 許金玉

國立臺灣大學/醫學院/臨床醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01183

摘要

試管嬰兒療程於刺激排卵及體外受精後，胚胎須在體外培養二至五天然後才植入子宮。培養的過程中每個胚胎發育的能力不一，有的胚胎生長良好，有些分裂速度較慢甚至停止分裂，有些則會出現細胞質的碎片。由於試管嬰兒療程的懷孕率有限，許多人工生殖機構為提高成功懷孕率，傾向植入數目較多的胚胎。而台灣對於試管嬰兒療程，醫療保險並未給付，患者對於懷孕成功的要求十分殷切，相對提高患者對多胞胎妊娠的忍受程度，造成後續高危險妊娠及早產兒的風險大為提高，從而耗費相當可觀的醫療資源。據此，本研究認為有必要針對分裂期(第三天)及囊胚期(第五天)胚胎植入的治療週期建立一套篩選標準，並提供依循這套選擇標準植入胚胎後，後續可能的懷孕率及多胞胎妊娠比率等相關數據，以便臨床醫師可以提供不孕夫妻適當的諮詢及建議。
本研究分別針對第三天胚胎及第五天囊胚植入的療程，以傳統胚胎型態選擇方法，分析其懷孕成功率及多胞胎妊娠率，結果發現第三天胚胎植入的療程，最佳三顆胚胎型態總分對於年齡小於35歲女性的族群，具有良好的能力以估計懷孕成功率及多胞胎妊娠率；但35歲以上女性的懷孕成功率，與胚胎型態就比較沒有關聯，反而與女性的年齡關係密切。對於囊胚療程來說，結果是類似的，只是對小於35歲的女性族群來說，最佳二顆及一顆囊胚的型態就可以分別估計療程懷孕成功率及多胞胎妊娠率；而最佳三顆囊胚型態的影響力可以擴充到介於35歲到38歲的女性；然而對於38 歲以上女性來說，仍須依賴女性的年齡以估計懷孕成功率。
上述的分析指出：胚胎品質對試管嬰兒療程懷孕成功率的影響甚鉅，而體外發育的胚胎，部分會停止分裂，這些胚胎被視為發生細胞老化的現象，且與粒線體內氧化自由基濃度有關。至於胚胎細胞出現碎片，則被認為是細胞凋亡的現象，並與細胞內氧化自由基濃度有關。胚胎分裂速度及胚胎碎片的多寡正好是胚胎型態的主要條件，因此本文接下來探討胚胎體外發育的微環境，也就是收集使用三天後的胚胎培養液，分析其中氧化自由基濃度與胚胎品質及著床能力的關聯性。結果發現：第三天胚胎培養液的氧化自由基濃度越高，則胚胎碎片程度越嚴重，且連帶胚胎的著床能力也下降。
胚胎細胞內的氧化自由基濃度與粒線體的代謝有關，而體細胞的研究中，一氧化氮具有調控粒線體呼吸的功能。我們先前的研究指出子宮內膜異位症患者，其異位的子宮內膜組織中一氧化氮合成酶表現增加，且一氧化氮的濃度升高，這可能是子宮內膜異位症造成不孕症的分子生物因素之一。此外，濾泡液中一氧化氮濃度升高，對於試管嬰兒療程第三天胚胎的品質有負面的影響。因此，後續以一個細胞期(合子)鼠胚體外發育過程作為動物模式，探討微環境中一氧化氮對於胚胎早期發育，尤其是粒線體功能的影響。
鼠胚實驗的結果發現，對照組的鼠胚在兩個細胞期胚胎的時候，會出現蛋白質巰基亞硝基化(S-nitrosylation)的狀況，且此時期巰基亞硝基化蛋白質與粒線體的位置相疊合。加入一氧化氮合成酶抑制劑之後，則會略微降低蛋白質巰基亞硝基化，然後讓鼠胚停留在兩個細胞期。倘若加入一氧化氮提供劑，則會造成巰基亞硝基化的程度提高，且逐漸出現胚胎碎片及細胞凋亡的現象。反之，若加入抗氧化劑如穀胱甘肽及類似超氧化物歧化酶的物質，則發現可以抵銷一氧化氮的負面作用，讓鼠胚得以繼續發育成為囊胚。其中，穀胱甘肽的效果最好，不但蛋白質巰基亞硝基化的程度恢復正常，且發育成的囊胚的腺苷三磷酸(ATP)的含量也正常。至於超氧化物歧化酶的效果則不是非常理想，因為蛋白質巰基亞硝基化的程度下降有限，並未恢復到正常程度，且囊胚的腺苷三磷酸的含量也較低。
總結來說，試管嬰兒療程體外受精後可得到一批批胚胎，同一批胚胎的發育能力並不相同。體外培養三到五天後，品質最佳的前三顆胚胎及女性年齡，可以用來指引醫師與患者夫婦協調植入胚胎的數目。而胚胎發育品質及後續的著床能力則與培養液中的氧化自由基濃度有關聯。鼠胚的實驗證實一氧化氮對於胚胎發育是必須的，但是太多量的一氧化氮反而會造成細胞碎片及凋亡，而加入抗氧化劑則有助於胚胎發育，這些作用機制都與粒線體的活性及蛋白質巰基亞硝基化有關。後續可再深入探討胚胎培養液中加入抗氧化劑是否可以改善胚胎品質並增進著床能力，進而提高試管嬰兒療程的懷孕成功率。

關鍵字

胚胎品質 ； 氧化自由基 ； 一氧化氮 ； 巰基亞硝基化 ； 細胞凋亡 ； 粒線體 ； 試管嬰兒療程

並列摘要

Subsequent to controlled ovarian stimulaiton and oocyte retrieval in assisted reproduction technology (ART) cycles, embryos are cultured in vitro of 2-5 days before transfer back into the uterus. During the process of embryonic development, some develop into good embryos, some features slow cleavage rates or arrest of development, while some appear cytoplasmic fragmentation. For the reason that the pregnancy rate in ART treatment is limited, many ART centers tend to transfer more number of embryos so as to enhance the pregnancy success rates. In Taiwan, since health insurance system does not pay for ART treatments, patients’ desire for pregnancy success is so strong that they consider multiple pregnancy acceptable. However, multiple gestation causes subsequent high risk pregnancy and prematurity, which may cost a great quantity of medical resources. Therefore, there is a need to establish a set of selection criteria for day 3 embryo transfer (D3ET) and blastocyst transfer (BT) cycles, so as to enable clinicians to provide infertile couples appropriate counseling.
This research analyzes the relevance of morphological parameters of embryo quality to the pregnancy success and multiple gestation in D3ET cycles or BT cycles. Among the common used criteria for embryo morphology, the embryo scores for the best three embryos (Top3) are the most crucial to predict the probability of pregnancy. Nevertheless, for women >35 years of age, it is female age that is the major factor for pregnancy outcome in D3ET cycles. After analysis of the BT program, the Top3 blastocyst scores can be utilized to decrease the number of embryos transferred in patients < 38 years of age. However, for women >38 years of age, it is female age acting the major factor for pregnancy outcome in BT cycles.
The above-mentioned analysis revealed that the effect of embryo quality on pregnancy outcome in ART cycles is prominent. The permanent arrest of embryo development is viewed as cellular senescence, which is connected with mitochondrial reactive oxygen species (ROS). Furthermore, cytoplasmic fragmentation of embryos is related to apoptosis, which is also connected with intracellular ROS levels. Therefore, the correlation between microenvironmental ROS levels and embryo quality were subsequently investigated. The results revealed that the ROS levels in spent culture media are correlated to embryo fragmentation degree. Furthermore, culture media ROS was inversely relevant to implantation potential of day 3 embryos.
The intracellular ROS levels of embryos are related to mitochondrial metabolism, which is regulated by nitric oxide (NO) in studies on somatic cells. Our previous reports indicated that, in patients with endometriosis, the expression of nitric oxide synthase increased in ectopic endometrial tissue. Furthermore, the elevated follicular NO levels are detrimental to embryo quality and may be related to failure of embryo implantation. Consequently, the in vitro development of mouse zygotes was utilized to elucidate the effect of microenvironmental NO on mitochondrial metabolism and blastocyst formation.
The results of mouse embryo experiment demonstrated that the apoptotic effect of NO on embryo development is closely related to protein S-nitrosylation within lipid-membrane rich organelles, such as mitochondria and endoplasmic reticulum. Totally abolishing NO production by NOS inhibitor is deleterious to embryo development and results in 2-cell block. The supplement of reduced form glutathione to NO-exposed embryos was able to maintain development competence, adequate proliferation of blastomeres, and ATP production in blastocysts.
In conclusion, the development potential is varied for the embryos in ART cycles. The best three embryos and the female age are both decisive to predict the pregnancy success and multiple gestation rates in D3ET and BT program, so that they could be utilized to help counseling for infertile couples and determine the number of transfer embryos. The fragmentation degree and implantation potential of embryos is correlated to the ROS levels in spent culture media. The mouse embryo experiment confirmed that NO is essential for embryo development. However, elevated microenvironmental NO levels may induce embryo degeneration and apoptosis. The addition of reduced glutathione is able to rescue the NO-exposed embryos. The effect of NO is intimately related to protein S-nitrosylation and mitochondrial activity. Further studies on the benefit of antioxidants in culture media on in vitro embryo development are deserved.

並列關鍵字

embryo quality ； reactive oxygen species ； nitric oxide ； S-nitrosylation ； apoptosis ； mitochondria ； assisted reproduction technology cycles

參考文獻

Acton BM, Jurisicova A, Jurisica I, Casper RF. Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development. Mol Hum Reprod. 2004 Jan; 10(1):23-32.

Agarwal A, Saleh RA, Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril. 2003 Apr; 79(4):829-843.

Agarwal A, Allamaneni SS, Said TM. Chemiluminescence technique for measuring reactive oxygen species. Reprod Biomed Online. 2004 Oct; 9(4):466-468.

Ahn HJ, Sohn IP, Kwon HC, Jo dH, Park YD, Min CK. Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos. Mol Reprod Dev. 2002 Apr; 61(4):466-476.

Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott RT. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril. 1999 May; 71(5):836-842.