胡晓东，熊风，万才云，李观贵，黄丽施，彭月婷，孙青，鲍忠剑，曾勇

(深圳中山泌尿外科医院生殖中心，深圳市围着床期生殖免疫重点实验室，深圳中山生殖与遗传研究所，深圳　518045)



·实验研究·

褪黑素对人卵裂期胚胎发育的影响及机制研究

胡晓东，熊风，万才云，李观贵，黄丽施，彭月婷，孙青，鲍忠剑，曾勇*

(深圳中山泌尿外科医院生殖中心，深圳市围着床期生殖免疫重点实验室，深圳中山生殖与遗传研究所，深圳518045)

目的探讨褪黑素对人第3天(D3)卵裂期胚胎发育成囊胚的影响及其作用机制。方法收集2012年10月至2014年10月在本中心行IVF-ET助孕治疗患者捐赠的D3玻璃化冷冻胚胎，复苏后存活率100%且未发生卵裂球溶解的胚胎纳入研究(n=143)，随机分为褪黑素组(培养液中加入1×10-7mol/L褪黑素，n=71)和对照组(不添加褪黑素，n=72)。两组胚胎均放于37℃、5%CO2培养箱中培养72 h，统计囊胚及优质囊胚形成率；采用鲁米诺化学发光法检测培养液中总活性氧(ROS)水平；实时荧光定量PCR法分析抗氧化酶类基因CAT、GPx、SOD1、SOD2及凋亡相关基因Bax、Bcl-2的表达。结果褪黑素组的囊胚形成率(42.25%)显著高于对照组(26.38%)(P<0.05)，优质囊胚形成率(30.00%)略高于对照组(26.32%)，但无显著性差异(P>0.05)。培养72 h后两组胚胎培养液中的ROS水平比较(513.33 vs. 556.67 RLUs)无显著性差异(P>0.05)。褪黑素处理后，胚胎内过氧化氢酶基因CAT的相对表达量(1.68±0.43)较对照组(1.00±0.03)显著上调(P<0.05)，其他基因包括Bax、Bcl-2、SOD1、SOD2、GPx的表达则没有显著性差异(P>0.05)。结论褪黑素能够促进人卵裂期胚胎继续发育形成囊胚，其机制可能与上调胚胎中过氧化氢酶基因CAT的表达有关。

褪黑素；人卵裂期胚胎；囊胚；过氧化氢酶

【Abstract】

Objective： To investigate the effect of melatonin on development of human cleavage-stage embryos to blastocyst in vitro.

Methods： The vitrified Day 3 embryos donated by couples undergone IVF treatment in our center from October 2012 to October 2014 were collected. The embryos which blastomere did not dissolved after warming were included in this study (n=143) and randomly divided into two groups according to the embryos were cultures with/without melatonin： the melatonin group (n=71) and the control group (n=72). The warmed embryos in both groups were cultured in the same incubator for 72 hours at 37℃，5% CO2concentration. The formation rates of blastocyst and high-grade blastocyst were compared between the two groups. Chemiluminescence assay was adopted to quantify the ROS levels in culture media. Gene expression of antioxidant enzymesCAT，GPx，SOD1 andSOD2，and apoptosis related genesBcl-2 andBaxin the embryos were analyzed by real-time fluorescent quantitative PCR.

Results： The blastocyst formation rate of melatonin group(42.25%)was significantly increased compared to the control group(26.38%)(P<0.05)，while the high-grade blastocyst formation rate was slightly increased but no statistic difference(30.00% vs. 26.32%)(P>0.05). The ROS levels in the culture media after 72 hours culture were comparable between the two groups(513.33 vs. 556.67 RLUs)(P>0.05). The mRNA level ofCATwas significantly increased in the melatonin group compared with the control group [(1.68±0.43)vs.(1.00±0.03)] (P<0.05)，but no significant difference was found in the mRNA level ofGPx，SOD1，SOD2，Bcl-2 andBax(P>0.05).

Conclusions： Melatonin can promote human Day 3 cleavage-stage embryos develop to blastocyst. The mechanism could be associated with up-regulating expression of catalase gene by melatonin.

(JReprodMed2016，25(8)：719-724)

胚胎发育发生在有氧环境中，因自身代谢会产生一定量的总活性氧(ROS)。RO……