Low tempature banking is now a routine component of ART (1). Cryopresvation refs to the storage of viable cells at low tempature (normal at -196℃). Cells are prone to damage during the cooling piod by three mechanisms: (a) chilling injury that occurs between +15 and -5℃, (b) ice crystal formation between-5 and℃, and (c) fracture damage i.e.,the mechanical effect of solidified fluid within the cell that occurs between-50 and-150℃(2). Storage at -196℃ is the least harmful aspect of the cryopresvation process. The cell has the potential to expience the same injury during the thawing process. Some strategies to prevent this damage include the avoidance of rapid tempature changes and decreasing the wat content of cells.

　　低溫保存是現(xiàn)在輔助生殖技術(shù)(ART)的常規(guī)組成部分(數(shù)據(jù)參考見1)。低溫保存是指在低溫(通常為-196°C)條件下保存活細(xì)胞。在冷卻期間，細(xì)胞容易受到三種機(jī)制的傷害:(a)溫度在15℃和-5℃之間的冷害，(B)-5℃和℃之間形成冰晶，和(C)斷裂損壞，即在-50℃和-150℃之間發(fā)生的細(xì)胞內(nèi)凝固液的機(jī)械效應(yīng)(數(shù)據(jù)參考見2)。低溫保存過程中危害較小的情況是在-196℃下儲存。細(xì)胞在解凍過程中可能遭受同等的損害。防止這種損害的一些策略包括避免溫度快速變化和降低細(xì)胞的水分含量。

　　Two basic techniques are employed for cooling of embryos: controlled slow freezing and ultra rapid vitrification (2). Both techniques utilize cryoprotective agents. Cryoprotective agents (CPAs) are used for long-tm presvation of ftilized embryos. CPAs like gcol are small, wat-soluble molecu that sve as antifreeze agents by disrupting hydrogen bonds between wat(1) Pmeable cryoprotectants prevent ice nucleation inside the cytoplasm ofthe cells, while extracellular cryoprotectants prevent ice crystal formation from the extracellular medium surrounding the cells. Non-pmeable cryoprotectants re on drawing wat from the cytoplasm through osmosis(2). Unfortunate, pmeable cryoprotectants have the potential to damage cells by direct advse effects and non-pmeable cryoprotectants may damage cells via osmotic effects. Cryoprotectant toxicity is proportional to its concentration and the duration of exposure(3).

　　冷卻胚胎有兩種基本技術(shù):受控緩慢保存和超快速玻璃化保存(數(shù)據(jù)參考見2)。兩種技術(shù)都使用了保存保護(hù)劑。保存保護(hù)劑(CPA)用于長期保存受精卵。保存保護(hù)劑例如丙三醇是小的水溶性分子，它們通過破壞水之間的氫鍵(數(shù)據(jù)參考見1)起到防凍劑的作用?？蓾B透的保存保護(hù)劑阻止細(xì)胞胞質(zhì)內(nèi)形成冰核，而細(xì)胞外的防凍劑可防止細(xì)胞周圍的胞外介質(zhì)形成冰晶。非滲透性保存保護(hù)劑通過滲透作用從細(xì)胞質(zhì)中吸取水分(數(shù)據(jù)參考見2)。不過，滲透性低溫保護(hù)劑可能有直接損害細(xì)胞的副作用損害細(xì)胞，而非滲透性低溫保護(hù)劑可能通過滲透作用損害細(xì)胞。低溫保護(hù)劑的毒性與其濃度和暴露時間成比例(數(shù)據(jù)參考見3)。

　　Slow freezing iolves cooling the cell at strict controlled slow rates, allowing wat to leave the cell aft treatment (1). Low concentrations of cryoprotectants are used in this process. Ultimate,this yields dense cytoplasmic viscosity without ice formation. Programmable freezing machines are used and freezing and thawing may take seval hours. The procedure also requires the use of expensive instrumentation. Cose,vitrification iolves rapid cooling of the cells. Transition from ambient tempature to -196°C occurs in s than a second. The cooling rate is above 15,000-30,000℃/minute (4). Cells treated with high doses of cryoprotectants are loaded into a carri and submged into liquid nitrogen. Vitrification essential solidifies the sample into a glass-like state,thus avoiding the formation of both intra-and extracellular Ice (5). While rapid cooling may prevent ice formation and chilling injury,the use of high doses of cryoprotectants may lead to direct injury. Vitrification is s expensive, as it does not iolve expensive instrumentation.Furthmore,this technique is more time-efficient,requiring on seval minutes as compared with 1-2 hours with controlled-rate slow freezing.

　　緩慢保存需以嚴(yán)格控制的緩慢速率冷卻細(xì)胞，使水在處理后脫離細(xì)胞(數(shù)據(jù)參考見1)。保存過程中會使用低濃度的低溫保護(hù)劑，較終會產(chǎn)生稠密的細(xì)胞質(zhì)粘度，而不會形成冰。這個過程中使用了可編程的保存機(jī)器，保存和解凍需要幾個小時的時間。該過程還需要使用昂貴的儀器。相反，玻璃化保存技術(shù)涉及細(xì)胞的快速冷卻。從周圍環(huán)境溫度到-196℃的轉(zhuǎn)變發(fā)生在不到一秒的時間內(nèi)。冷卻速度為15,000-30,000℃/分鐘以上(數(shù)據(jù)參考見4)。將經(jīng)更高劑量低溫保護(hù)劑處理過的細(xì)胞裝載到載體中，然后浸入到液氮中。玻璃化保存基本上將樣品固化成玻璃狀，從而避免了細(xì)胞內(nèi)外冰晶(數(shù)據(jù)參考見5)的形成。雖然快速冷卻可以防止冰的形成和冷害，但是使用更高劑量的低溫保護(hù)劑可能會帶來直接傷害。玻璃化保存的成本較低，因?yàn)樗簧婕鞍嘿F的儀器。此外，該技術(shù)更省時，只需要幾分鐘，而控制速率的緩慢保存卻需要1-2小時。

　　Kolibianakis and colleagues recent undtook a meta-anasis of randomized trials comparing slow freezing to vitrification (6). They found that vitrification yielded high post thaw survival rates for cleavage stage embryos (odds ratio (OR): 6.35,95% confidence intval(CI):1.14-35.26) and blastocysts(OR: 4.09,95% CI:2.45-6.84). Howev, ovall pregcy rates did not diff between the two groups (OR:1.66,95% CI:0.98-2.79). Hence,the method of embryo freezing used should depend on the exptise of each ART laboratory.

　　Kolibianakis及其同事較近對緩慢保存和玻璃化保存對比的隨機(jī)試驗(yàn)進(jìn)行了元分析(數(shù)據(jù)參考見6)。他們發(fā)現(xiàn)，對于卵裂期胚胎(優(yōu)勢比(OR):6.35，95%可信區(qū)間(CI):1.14-35.26)和囊胚(OR: 4.09，95% CI: 2.45-6.84)來說，玻璃化解凍后的胚胎存活率更高。然而，兩組的總?cè)焉锫什o差異(OR :1.66，95%可信區(qū)間:098-2.79)。因此，使用何種胚胎保存方法應(yīng)取決于每個ART實(shí)驗(yàn)室的專業(yè)能力。

　　Refence 數(shù)據(jù)參考：

　　1. Gosden R. Cryopresvation: a cold look at technology for ftility

　　presvation. Ftil Stil. 2011; 96 (2) : 264-8.

　　2. Ata B, Chian RC, Tan SL. Cryopresvation of oocytes and embryos for

　　ftility presvation for female canc patients. Best Pract Res Clin

　　Obstet Gynaecol. 2010; 24 (1) : 101-12.

　　3. Vajta G, Kuwayama M, Vandzwalmen P. Disadvantages and benefits

　　of vitrification. In Tuck MJ & Liebmann J (Eds.). Vitrification in

　　assisted reproduction A us's manual and troubhooting guide.

　　London: Informa UK, 2007,pp.33-44.

　　4. Liebmann J, Nawroth F, Isachenko V,et al. Potential importance of

　　vitrification in reproductive medicine. Biol Reprod. 2002; 67: 1671.

　　5. Vajta G, Kuwayama M. Improving cryopresvation systems.

　　Thiogenology. 2006; 65: 236-44.

　　6. Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopresvation of human

　　embryos by vitrification or slow freezing: which one is bett? Curr

　　Opin Obstet Gynecol. 2009; 21 (3) : 270-4.

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