胚胎冷冻
Low temperature banking is now a routine component of ART (1). Cryopreservation refers to the storage of viable cells at low temperature (normally at -196℃). Cells are prone to damage during the cooling period by three mechanisms: (a) chilling injury that occurs between +15 and -5℃, (b) ice crystal formation between-5 and-80℃, 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 cryopreservation process. The cell has the potential to experience the same injury during the thawing process. Some strategies to prevent this damage include the avoidance of rapid temperature changes and decreasing the water content of cells.
低温保存是现在辅助生殖技术(ART)的常规组成部分(数据参考见1)。低温保存是指在低温(通常为-196°C)条件下保存活细胞。在冷却期间，细胞容易受到三种机制的伤害:(a)温度在15℃和-5℃之间的冷害，(B)-5℃和-80℃之间形成冰晶，和(C)断裂损坏，即在-50℃和-150℃之间发生的细胞内凝固液的机械效应(数据参考见2)。低温保存过程中危害最小的情况是在-196℃下储存。细胞在解冻过程中可能遭受同等的损害。防止这种损害的一些策略包括避免温度快速变化和降低细胞的水分含量。
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-term preservation of fertilized embryos. CPAs like glycerol are small, water-soluble molecules that serve as antifreeze agents by disrupting hydrogen bonds between water(1) Permeable cryoprotectants prevent ice nucleation inside the cytoplasm ofthe cells, while extracellular cryoprotectants prevent ice crystal formation from the extracellular medium surrounding the cells. Non-permeable cryoprotectants rely on drawing water from the cytoplasm through osmosis(2). Unfortunately, permeable cryoprotectants have the potential to damage cells by direct adverse effects and non-permeable cryoprotectants may damage cells via osmotic effects. Cryoprotectant toxicity is proportional to its concentration and the duration of exposure(3).
冷却胚胎有两种基本技术:受控缓慢冷冻和超快速玻璃化冷冻(数据参考见2)。两种技术都使用了冷冻保护剂。冷冻保护剂(CPA)用于长期保存受精卵。冷冻保护剂例如丙三醇是小的水溶性分子，它们通过破坏水之间的氢键(数据参考见1)起到防冻剂的作用。可渗透的冷冻保护剂阻止细胞胞质内形成冰核，而细胞外的防冻剂可防止细胞周围的胞外介质形成冰晶。非渗透性冷冻保护剂通过渗透作用从细胞质中吸取水分(数据参考见2)。不过，渗透性低温保护剂可能有直接损害细胞的副作用损害细胞，而非渗透性低温保护剂可能通过渗透作用损害细胞。低温保护剂的毒性与其浓度和暴露时间成比例(数据参考见3)。
Slow freezing involves cooling the cell at strictly controlled slow rates, allowing water to leave the cell after treatment (1). Low concentrations of cryoprotectants are used in this process. Ultimately,this yields dense cytoplasmic viscosity without ice formation. Programmable freezing machines are used and freezing and thawing may take several hours. The procedure also requires the use of expensive instrumentation. Conversely,vitrification involves rapid cooling of the cells. Transition from ambient temperature to -196°C occurs in less than a second. The cooling rate is above 15,000-30,000℃/minute (4). Cells treated with higher doses of cryoprotectants are loaded into a carrier and submerged into liquid nitrogen. Vitrification essentially 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 higher doses of cryoprotectants may lead to direct injury. Vitrification is less expensive, as it does not involve expensive instrumentation.Furthermore,this technique is more time-efficient,requiring only several minutes as compared with 1-2 hours with controlled-rate slow freezing.
缓慢冷冻需以严格控制的缓慢速率冷却细胞，使水在处理后脱离细胞(数据参考见1)。冷冻过程中会使用低浓度的低温保护剂，最终会产生稠密的细胞质粘度，而不会形成冰。这个过程中使用了可编程的冷冻机器，冷冻和解冻需要几个小时的时间。该过程还需要使用昂贵的仪器。相反，玻璃化冷冻技术涉及细胞的快速冷却。从周围环境温度到-196℃的转变发生在不到一秒的时间内。冷却速度为15,000-30,000℃/分钟以上(数据参考见4)。将经更高剂量低温保护剂处理过的细胞装载到载体中，然后浸入到液氮中。玻璃化冷冻基本上将样品固化成玻璃状，从而避免了细胞内外冰晶(数据参考见5)的形成。虽然快速冷却可以防止冰的形成和冷害，但是使用更高剂量的低温保护剂可能会带来直接伤害。玻璃化冷冻的成本较低，因为它不涉及昂贵的仪器。此外，该技术更省时，只需要几分钟，而控制速率的缓慢冷冻却需要1-2小时。
Kolibianakis and colleagues recently undertook a meta-analysis of randomized trials comparing slow freezing to vitrification (6). They found that vitrification yielded higher post thaw survival rates for cleavage stage embryos (odds ratio (OR): 6.35,95% confidence interval(CI):1.14-35.26) and blastocysts(OR: 4.09,95% CI:2.45-6.84). However, overall pregnancy rates did not differ between the two groups (OR:1.66,95% CI:0.98-2.79). Hence,the method of embryo freezing used should depend on the expertise of each ART laboratory.
Kolibianakis及其同事最近对缓慢冷冻和玻璃化冷冻对比的随机试验进行了元分析(数据参考见6)。他们发现，对于卵裂期胚胎(优势比(OR):6.35，95%可信区间(CI):1.14-35.26)和囊胚(OR: 4.09，95% CI: 2.45-6.84)来说，玻璃化解冻后的胚胎存活率更高。然而，两组的总妊娠率并无差异(OR :1.66，95%可信区间:098-2.79)。因此，使用何种胚胎冷冻方法应取决于每个ART实验室的专业能力。
Reference 数据参考：
1. Gosden R. Cryopreservation: a cold look at technology for fertility
preservation. Fertil Steril. 2011; 96 (2) : 264-8.
2. Ata B, Chian RC, Tan SL. Cryopreservation of oocytes and embryos for
fertility preservation for female cancer patients. Best Pract Res Clin
Obstet Gynaecol. 2010; 24 (1) : 101-12.
3. Vajta G, Kuwayama M, Vanderzwalmen P. Disadvantages and benefits
of vitrification. In Tucker MJ & Liebermann J (Eds.). Vitrification in
assisted reproduction A user's manual and troubleshooting guide.
London: Informa UK, 2007,pp.33-44.
4. Liebermann J, Nawroth F, Isachenko V,et al. Potential importance of
vitrification in reproductive medicine. Biol Reprod. 2002; 67: 1671-80.
5. Vajta G, Kuwayama M. Improving cryopreservation systems.
Theriogenology. 2006; 65: 236-44.
6. Kolibianakis EM, Venetis CA, Tarlatzis BC. Cryopreservation of human
embryos by vitrification or slow freezing: which one is better? Curr
Opin Obstet Gynecol. 2009; 21 (3) : 270-4.
