Objective: To study the amino acid metabolism of human early frozen-thawed embryo.Methods: Eighteen spare human embryos obtained from 13 patients undergoing in vitro ferbilization (IVF) were researched.Spare human embryos on day 3 of development were cultured individually in 20 μl drops of pre-equilibrated blastocyst culture medium for 2 hours before vitrification.Embryo-free drops were incubated in the same dish as the controls.The remaining 15 μl mediums from the drops were collected before freezing, 1/2,1,2,4,6 and 24 hours after thawing, and were analyzed for 20 free amino acids level by high performance liquid chromatography ( HPLC).Results: The levels of glutamine, histidine, tryptophan and lysine in blank controls were different among different time points.However, they were not increased or decreased gradually.The concentrations of other 16 amino acids remained same at different time points.One hour after thawing, concentrations of 20 free amino acids were all increased comparing with the blank control at same time point; the amino acid appearance and turnover was significant higher than that pre-freezing ( P ＜ 0.05).The amino acid appearance had no significant difference betweem 2, 6 or 24 hours after thawing and pre-freezing ( P ＞ 0.05).The amino acid depletion pre-freezing was significant lower than that 1/2, 4, 6 and 24 hours after thawing (P ＜0.05).The amino acid appearance 1/2 and 4 hours after thawing was significant lower than that pre-freezing ( P ＜ 0.05).There was no significant difference in amino acid turnover between 1/2,4 hours after thawing and pre-freezing (P ＞ 0.05).The amino acid turnover 24 hours after thawing was significant higher than that pre-freezing (P ＜ 0.05).The amino acid turnover 1/2,4 and 6 hours after thawing were significant lower than that 1 hour after thawing (P ＜0.05).Conclusion: Human early embryo begins amino acid metabolism and recovers from metabolism stasis 1/2 hour after embryo thawing , and the amino acid metabolism of the embryo at this time is close to that pre-freezing.%目的:通过检测冻融胚胎解冻后的氨基酸代谢水平,研究人类早期胚胎解冻后的氨基酸代谢规律,为确定冻融胚胎解冻后的适宜移植时机提供依据.方法:选择2010年9-12月接受体外受精-胚胎移植治疗的13例患者共18个取卵后第3日6~8细胞废弃胚胎,20 μl囊胚培养液微滴中单独培养2 h后行玻璃化冷冻保存,分别于冷冻前2 h、解冻后1/2、1、2、4、6、24 h收集胚胎培养液微滴15 μl,同一培养皿中未培养胚胎的培养液为空白对照组.采用高效液相色谱法(HPLC)检测标本中20种游离氨基酸浓度.观察胚胎冷冻前、解冻后不同时间点氨基酸代谢的变化.结果:HPLC检测发现,空白对照组培养液中谷氨酰胺、组氨酸、色氨酸及赖氨酸在不同时间点的浓度变化有统计学意义,但是没有随培养时间的延长而逐渐增高或降低;其余16种氨基酸在冷冻前和解冻后不同时间点的浓度基本保持不变.解冻后1 h胚胎培养液中20种游离氨基酸浓度较相应时间点的空白对照组增加,且氨基酸增加量和氨基酸转换量高于冷冻前(P<0.05);解冻后2 h胚胎培养液中20种氨基酸浓度也较空白对照组增加,解冻后2、6及24 h氨基酸增加量和冷冻前比较差异均无统计学意义(P>0.05);解冻后1/2、4、6及24 h胚胎培养液中氨基酸减少量高于冷冻前(P<0.05);解冻后1/2、4 h胚胎培养液中氨基酸增加量低于冷冻前(P<0.05),氨基酸转换量也低于冷冻前,但差异无统计学意义(P>0.05);解冻后24 h胚胎培养液中氨基酸转换量高于冷冻前(P<0.05);解冻后1/2、4及6 h胚胎培养液中氨基酸转换量低于解冻后1 h(P<0.05).结论:人类早期胚胎解冻后1/2 h已经从冷冻时的代谢停滞状态恢复,开始了氨基酸代谢,此时氨基酸代谢水平已接近于胚胎冷冻前的状态.
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