Effect Of Melatonin On Attenuating Impaired Development Of Aging Oocytes After In Vitro Fertilization

Older women receiving in vitro fertilization?IVF?treatment always have significantly lower pregnancy outcome.Among the potential reasons,impaired embryonic development induced by aging oocytes is thought to contribute largely to the lower pregnancy rate and higher miscarriage rate.Therefore,it is significantly important to find a practical methodology that can attenuate the impaired development of the aging oocytes after IVF.In this study,mice oocytes are used as the research object,and the model of stress-induced premature senescence?SIPS?is induced by oxidative stress?H₂O₂?in vitro to study the effects of oxidative stress on aging oocytes cause of mitochondria-related characteristics of damage,whether it is important reasons for leading to IVF embryonic development and quality decline.Finally,we improve the developmental rate of blastocyst and the quality of blastocysts by adding the concentration of melatonin in vitro culture medium.This study explore the possibility of melatonin to improve the embryonic development and quality of aging oocytes and reveale its possible mechanism,to provides a novel strategy for enhancing the success rate of IVF in clinical elderly women.In the present study,fresh MII oocytes were treated with 10 ?M,50 ?M,100 ?M and 150 ?M H₂O₂ for 3 hours respectively,to establish the aging oocyte model characterized with oxidative stress.The oocytes without H₂O₂ treatment were used as controls.The development rate in each group was detected after standard IVF.Our results showed that the fragmentation rate at 2-cell stage after 150 ?M H₂O₂ treatment was significantly higher than that of the control group?7.77±4.78% vs.39.32±9.14%,P < 0.05?.The cleavage rate after 100?M H₂O₂ treatment was significantly lower?45.63 ± 2.6%?than that of control group?54.67±8.27%;P < 0.05?.At the blastocyst stage,50 ?M,100 ?M H₂O₂ treated oocytes showed significantly lower blastocyst rates?26.27 ± 0.06%,28.46 ± 3.45%,respectively?than that in the control group?34.9 ± 1.77%,P < 0.05?.These results indicated that the oxidative stress accumulated in aging oocytes can continuously damage the embryonic development after IVF.In many cellular types,aging is always associated with mitochondrial dysfunctions.Thus,we detect mitochondrial mass and functions of H₂O₂ induced-aged oocytes.The active mitochondria detected by Mitotracker Red showed an evident decrease in active mitochondrial mass of aging oocytes.Similarly,by quantitatively detecting the mitochondrial DNA?mtDNA?copy number,we showed that the mtDNA copy number of aging oocytes tend to decrease with the increased H₂O₂ concentration.In addition,JC-1 probe detection showed that the mitochondrial membrane potential?MMP?of aging oocytes appear to be increased.This phenomenon is termed as hyperpolarization,which is a very early event of apoptosis.This implies that apoptosis is likely to occur in aging oocytes.The continuous development impairment implies that the damage induced by oocyte aging can be reversed.Thus,we supplement melatonin?MT?in culture medium of IVF embryos,to test if the impaired IVF development can be attenuated by scavenging reactive oxygen species?ROS?accumulated in preimplantation embryos.Using aging oocytes induced by 100 ?M H₂O₂,a relative mild conditions,we supplement different concentrations?10-5 M,10-7 M,10-9 M?of melatonin to the culture medium IVF.Compared with that in control aging group,10-9 M melatonin supplementation could significantly increase the blastocyst rate?29.42 ± 2.39% vs 20.47 ± 4.11%?,which was comparable to the development rate control in young oocytes group?38.76 ± 9.43%?.While the 10-5 M and 10-7 M supplmentation did not improve the blastocyst rate?19.87 ± 2.88% and 23.35 ± 6.03%?.These results suggest that melatonin supplementation at low concentration in culture medium can effectively reverse the developmental potential of aging oocytes.Next,we found that melatonin supplementation at different concentrations?10-5 M?10-7 M?10-9 M?did not affect the cell number of blastocysts?28.30±10.42%?31.54±9.97%?39.36±9.78%,respectively?,compared with that from aging oocytes?37.91 ± 4.25%?.Furthermore,we showed 10-9 M melatonin supplementation appear to inhibit apoptotic rate?2.57% vs.3.18%,P > 0.05?.Unexpectedly,we found that 10-5 M significantly increased apoptotic rate in blastocyst?6.97% vs.3.18%,P <0.05?,implying that high concentration melatonin had an adverse effect on the IVF development of aging oocytes.Moreover,the quantitation of DNA copy number showed that 10-9 M melatonin supplementation significantly reversed mtDNA loss in IVF embryos from aging oocytes at 8-cell stage.CONCLUSION:1)In the aging oocytes induced by 100 ?M H₂O₂,intra-oocyte ROS levels significantly increased,accompanied with mitochondrial dysfunctions and impairment,including decreased active mitochondrial mass and mtDNA copy number,mitochondrial membrane hyperpolarization.2)Supplementation of 10-9 M melatonin into the culture medium,significantly reversed the decrease in mtDNA copy number induced by H₂O₂ treatment,thus attenuate the developmental arrest of aging oocytes and improve the developmental rate and quality of blastocyst after in vitro fertilization.