长时受精过程中活性氧及精子的DNA损伤对体外受精结局的影响

Impact of ROS and sperm DNA damage on the outcome of in vitro fertilization under long-term fertilization

目的通过检测精液处理后不同时间的精子DNA损伤程度与活性氧的变化,分析对比长时受精与短时受精两种精卵孵育时间的实验室参数及临床结局,旨在探讨长时受精对体外受精-胚胎移植治疗中的不利影响.方法接受常规IVF受精的夫妇146例,共取卵146个周期,移植113个周期.在取卵当日采集精液,经处理后将精子浓度调整为10×106/ml,留取标本3份,各1.0ml.第一份标本定为加精后0h（即在CO2培养箱内培养至该患者卵子加精时检测）,第二份标本定为加精后5h（即在CO2培养箱内培养至该患者卵子加精后5h后检测）,第三份标本定为加精后20h（即在CO2培养箱内培养至该患者卵子加精后20h后检测）,每份标本均用于检测H2O2、CTA和精子DNA损伤.精子DNA损伤采用精子吖啶橙染色方法测定,H2O2及CTA含量测定采用分光光度比色测定法.在胚胎移植日,除外取消移植的患者33名,其余的113名患者随机分成两组,A组57人选择短时受精的胚胎进行移植,B组56人选择长时受精的胚胎进行移植.结果精子DNA损伤、H2O2水平在加精后20h均明显高于授精时及授精后5h（P〈0.05）,CTA水平明显低于授精时及授精后5h（P〈0.05）.精子DNA损伤率在授精后三个时间均与H2O2浓度呈正相关（r值分别为0.688、0.532和0.491, P〈0.05）;精子DNA损伤率与授精后20h的CTA浓度呈负相关（r=-0.347, P〈0.05）.B组的多精受精率明显高于A组（P〈0.05）,A组的优胚率明显高于B组（P〈0.05）,两组间的受精率、卵裂率、植入率、临床妊娠率无统计学差异.结论长时受精时,精子可以产生过量的活性氧并且导致DNA损伤的精子增多,对卵子及胚胎有不利影响.短时受精在不影响受精率及卵裂率的前提下,可以降低多精受精率,提高优胚率.

Objective To detect the change of sperm DNA damage and reactive oxygen species at different time after insemination and explore the adverse effects of long-term fertilization on outcome of in vitro fertilization by comparative analysis of the laboratory and clinical data of short-term fertilization and long-term fertilization. Methods One hundred and forty-six patients who underwent in vitro fertilization and embryo transfer for female infertility were enrolled in the study. Semen was collected on the oocyte retrieval day and treated with Pure Sperm. Three samples were taken after the sperm concentration adjusted to 10×106/ml and the volume of each sample was 1.0ml. The first sample was defined as 0h after insemination （when oocytes added sperm）, the second sample was defined as 5h after insemination（5h after oocytes added sperm） and the third sample was defined as 20h after insemination（20h after oocytes added sperm）. Each sample was used for detecting of H2O2, CTA and sperm DNA damage. Sperm DNA damage levels were determined with acridine orange staining. The concentrations of hydrogen peroxide and catalase were detected by colorimetric. On the embryo transfer day, the patients were divided into two groups： one group of patients chose short-term fertilized embryo to transfer, one group of patients chose long fertilized embryo to transfer. Results The levels of sperm DNA damage and hydrogen peroxide in the sperm wash medium at 20h after insemination were higher than those at 0h and 5h after insemination significantly （P〈0.05）, and the levels of catalase were lower than that at 0h and 5h after insemination significantly （P〈0.05）. The sperm DNA damage levels at 0h, 5h, 20h after insemination were positively correlated with the concentrations of hydrogen peroxide（r=0.688, 0.532, 0.491, P〈0.05）. The sperm DNA damage levels were negatively correlated with the concentrations of catalase at 20h after insemination（r=-0.347, P〈0.05）. The polyspermy rate in group B was significantly higher than that in group A （P〈0.05）, and the high quality embryo rate in group A was significantly higher than that in group B（P〈0.05）. No significant differences were found in fertilization rate, cleavage rate, implantation rate, clinical pregnancy rate between the two groups （P〉0.05）. Conclusion For long-term fertilization, the sperm could produce excessive reactive oxygen and enhanced sperm DNA damage,which led to have an adverse effect on the oocytes and embryos. Short-term fertilization could reduce the polyspermy rate and improve the high quality embryo rate under no effecton the fertilization rate and cleavage rate.