精蟲品質及著床前期胚胎發育與冷凍保存之研究

第一部份具有完整去氧核醣核酸的精蟲才能傳遞正確的遺傳訊息給下一代，而受精成功是生命誕生的第一步。本篇實驗目是要鑑定精蟲去氧核醣核酸片段化對臨床上精蟲品質與試管嬰兒療程之影響。實驗發現精蟲異常病患有較高之精蟲去氧核醣核酸片段化發生率，當精蟲去氧核醣核酸片段化大於10% 時其試管嬰兒受精率會受影響。精蟲去氧核醣核酸片段化與精蟲速率呈負相關，但不影響懷孕率。在施行試管嬰兒療程之前，如何篩選品質優良精蟲就成為重要課題。第二部份前列環素是一種激素類物質，產生於前列腺，具有抗凝血和擴張血管的作用。最近科學家的研究指出人類輸卵管細胞會合成大量前列環素，而胚胎早期發育的前72小時是在輸卵管內，故本實驗以鼠胚培養於含前列環素類似物的培養液，評估前列環素對胚胎早期發育的影響。結果發現前列環素在四細胞到桑椹胚期會幫助胚胎早期發育和孵化，在人類輸卵管培養液含1%牛血清白蛋白時每毫升1.0微莫耳前列環素類似物會顯著提高胚胎早期發育和孵化率，在單純人類輸卵管培養液時每毫升2.0微莫耳前列環素類似物亦會顯著提高胚胎早期發育和孵化率，但已孵化之囊胚總細胞數和囊胚體積則無顯著差異。第三部份雖然卵子及胚胎冷凍方法研究不斷被開發，然而胚胎在不同的發育階段，究竟適用於何種冷凍方法是最有利的?依冷凍原理分類，主要有(1)傳統慢速冷凍-平衡冷凍，及降溫過程冷凍保護劑與細胞仍不斷交互作用以達平衡，(2)急速玻璃化冷凍-為非平衡冷凍，因為高濃度的冷凍保護劑處理，再短時間內可達平衡，這種平衡現象在冷凍之前就已完成，且降溫速率快速，細胞瞬間玻璃化而沒有冰晶的形成，傳統的慢速冷凍對於分裂期的胚胎，近來在部份實驗室中已被急速玻璃化冷凍所取代，本實驗目的在於探討不同的凍保存方法，對於不同時期胚胎包括受精合子(zygotes)及囊胚(blastocysts)冷凍之影響，實驗材料包括以小鼠及捐贈之人類胚胎作為測試，並進一步評估臨床之結果，冷凍方法包括(1)傳統慢速冷凍，及一種相較於急速玻璃化冷凍之冷凍速率更快的方法-(2)超高速玻璃化急速冷凍法，來評估此兩種方法之結果，以及是否有較好應用於臨床的結果，而超高速玻璃化急速冷凍法是利用儀器Vit-Master以減壓降溫的方法，先將液態氮降溫到-205至-210℃，再將胚胎急速降溫，在小鼠的實驗當中，實驗結果証實，小鼠之受精合子及囊胚可以成功的以超高速玻璃化急速冷凍法保存，並且於解凍後有高達87-91%的存活率，及50-56%之孵化率。利用試管嬰兒中經由病人所捐贈之剩餘囊胚，予以超高速玻璃化急速冷凍，結果發現冷凍解凍後的胚胎存活率高達86%。進一步應用於臨床試管嬰兒病人，將剩餘胚胎繼續培養所得之囊胚，予以超高速玻璃化急速冷凍保存，在解凍後予以植入病人後，發現其懷孕率為47.5%，更相較於傳統慢速冷凍法之結果(懷孕率30%)更為良好。而人類受精合子以慢速冷凍之臨床結果比超急速玻璃冷凍來的好，比起慢速冷凍法冰凍囊胚期胚胎，超高速玻璃化急速冷凍法，不僅操作簡單而且效率更佳，其解凍存活率比慢速冷凍法更好，因此懷孕率也更高。

關鍵字

去氧核醣核酸片段化；前列環素；冷凍胚胎

並列摘要

part 1 Sperm DNA integrity is essential for accurate transmission of genetic material to offsprings. Fertilization marks the creation of a new and unique individual. The aim of this study was to determine the incidence of sperm nuclear DNA fragmentation, to correlate these results with semen analysis parameters and IVF outcomes. The sperm DNA fragmentation rate of abnormal sperm patients are significantly higher than those of the normal sperm patients. And, the fertilization rates were affected in sperm DNA fragmentation > 10% patients. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes. Before IVF, the selection of good quality sperm for oocyte is very important. part2 Because human fallopian tube cells synthesize abundant amounts of prostacyclin (PGI2). And the first 72 hours of embryo development take place in the oviduct. We cultured mouse embryos with a PGI2 analogue to evaluate in vitro effects. Exposure to PGI2 analogue during the four-cell to morula stages was critical to enhanced embryo development and hatching but did not increase blastocyst volume or cell number of hatched blastocysts. The effects of PGI2 analogue were statistically significant at 1.0 µmol/L and 2.0 µmol/L in human tubal fluid (HTF) medium with or without 1% bovine serum albumin. We found that PGI2 analogue enhanced embryo development and hatching, but PGI2 did not increase number of cells in hatched blastocysts or blastocyst volume. part 3 Although the cryopreservation methods of oocytes and embryos have continually been developed, which methods are best for embryos storage at different development stage is unknown. The cryopreservation methods could be divided to classical slow cooling and vitrification according by the principle of cryobiology. the slow cooling cryopreservation is a “equilibrium cooling” freezing and the interaction between cryoprotectants and cell is continually occurred in duration of reducing temperature; the vitrification is a “non-equilibrium cooling”, because the balance between highe concentration cryoprotectants and cells is completely finish at short time of treatment before temperature reducing. The cooling rate of vitrification is rapid and liquid phase turns to glass phase without ice cryostal formation. The classical slow cooling method is replaced by the vitrification on preimplantation embryos in several labotary, recently. The purpose of this study is to assess the effects of zygotes and blastocysts cryopreservation by two different freezing methods. Mouse and donated human embryos are used for the experiments and then further assess the clinical results on iv vitro fertilization. The cryopreservations on this study are including (1) classical slow cooling and (2) super-ultra rapid vitrification, an ultra rapid cooling rate than vitrification to assess the results on IVF. Ultra rapid cooling rate of the super-ultra rapid vitrification is made by the mechiane- Vitmaster to decrese pressure by suction and the tempature of liquid nitrogen is reduced to -205 ~ -210℃. The embryos after treating with cryoprotectants are loading in to the liquid nitrogen with -205~ -210℃ to achived a glass phase with ultra rapid cooling rate. In the results of mouse embryo cryopreservation, zygotes and blastocysts can be successful freezing by super-ultra vitrification and the survival and hatching rates are around 87-91% and 50-56%, respiretively. The super-ultra rapid vitrafication experiment of human blastocysts donated from patients underwent IVF program has a suessfull survival rate (86%). Furthermore, the pregnancy rate on patients transferred with warmed blastocysts cryopreserved with super-ultra rapid vitrification is 47.5% and the result is better than that with classical slow cooling method (30%). On the other hand, the clinical pregnancy rate on the group of zygotes with slow cooling is successful than that with super ultra vitrification. The super-ultra rapid vitrification is a simple method and has a higher survival and pregnancy rate on blastocysts freezing than slow cooling.

並列關鍵字

TUNEL ； Prostacyclin ； cryopreservation

參考文獻

Angelopoulos T, Moshel YA, Lu L, Macanas E, Grifo JA and Krey LC 1998 Simultaneous assessment of sperm chromatin condensation and morphology before and after separation procedures: effect on the clinical outcome after in vitro fertilization. Fertil Steril, 69, 740-747.

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Bianchi PG, Manicardi D, Bizzaro D, Bianchi U and Sakkas D. 1993. Effect of deoxyribonucleic acid protamination on fluorochrome staining and in situ nick-translation of murine and human spermatozoa. Biol Reprod, 49, 1083-1088.

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Bonduelle M, Aytoz A, Van Assche E, Devroey P, Liebaers I, Van Steirteghem A 1998. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum Reprod, 13, 781-782.

國際替代計量

精蟲品質及著床前期胚胎發育與冷凍保存之研究

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