| Objective 1.This study was performed to research the effect of melatonin on human oocyte in vitro maturation and subsequent embryonic development, then selecting the optimum melatonin concentration in maturation culture medium.2. According to the differences in the mitochondrial membrane potential of oocytes between experimental group and control group, the mechanism of melatonin act on immature oocytes in vitro maturation are explored.Methods 1.A total of 602 immature oocytes from stimulated intracytoplasmic sperm injection（ICSI） cycles were randomly divided into six groups and cultured individually in maturation medium containing different concentrations of melatonin(0,10-11,10-9,10-7,10-5,10-3 M).The group without melatonin was control group,the others were experimental groups.After 24 hours, the oocytes with a polar body were regarded as mature. The mature oocytes are injected with ICSI, about 16-18 h after injection, we can observe the pronuclei to judge fertilization.The fertilized egg are cultured to blastocyst.Oocytes maturation, fertilization, cleavage, blastocyst and good quality blastocyst rates were recorded.2. The mitochondrial membrane potential of 15 mature oocytes from the 10-5 group and 13 mature oocytes from the control group are dyed by JC-1. The red/blue fluorescent emissions from the oocytes were recorded as tagged image format files using fluorescence microscope. The recorded red/blue fluorescent images were analyzed using image software（v. 1.40） by mean gray values of fluorescent,then the ratio of red fluorescence intensity and green fluorescence intensity were calculated to assess the mitochondrial function.Results 1. The results showed that the good-quality blastocyst rate were significantly higher in the group supplemented with 10-9M,10-7M,10-5M melatonin compared with the control group（75±4.4%,63.5±5.4%,60±4.7% VS 11.1±5.4%,p<0.05）.However,the cleavage rate and the blastocyst rate were significantly lower in the 10-3 M melatonin treatment than those in the control group（73.8±1.2%VS 90.2±0.6%;2±0.2%VS 16.4±0.6%;p<0.05）.in this study,a total of 54 blastocysts were formed, including 30 good-quality blastocysts. 11 good-quality blastocysts（10 from the experimental groups and 1 from control group） were selected randomly and thawed for the detection of aneuploidy through a CGH.The embryo from the control group was diagnosed as aneuploid [49,XX（+1,+15,+17）],whereas of the ten from the experimental groups, one was aneuploid[46,XX（+11,-15）](from melatonin of 10-11); one has a 4.75 Mb losses of 7（q36.236.3）(from melatonin of 10-7) and the other eight were normal(4 from melatonin of 10-9,1 from 10-7,2 from 10-5,1from 10-11).2. The inner membrane potential in MII oocytes from melatonin in 10-5 M group were higher than those in non-supplemented control group（0.58±0.3 vs.0.35±0.17,p<0.05）.Conclusions 1. melatonin in proper concentration added in maturation medium could improve human oocyte developmental competence in IVM.When the melatonin is in 10-9mol/l,we could get more normal good quality blastocyst.2. Appropriate concentration of melatonin could improve the quality of oocyte in vitro maturation. |
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