邻苯二甲酸二丁酯暴露对不孕女性助孕结局及小鼠早期胚胎发育影响的研究

Effects of exposure to di-n-butyl phthalate on pregnancy outcome in infertile women and early embryo development in mice

目的探讨体内邻苯二甲酸二丁酯(di-n-butyl phthalate,DBP)暴露水平对不孕患者早期助孕结局的影响及其对小鼠早期胚胎发育的影响。方法本研究选取2019年2月至10月期间在河南省人民医院生殖中心就诊的因输卵管因素行体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)治疗的患者,根据病历号尾号抽取单号的112例不孕患者为研究对象,应用高效液相色谱串联质谱检测尿液中DBP代谢产物邻苯二甲酸单丁酯(mono-n-butyl phthalate,MBP)的浓度,采用多元回归模型分析尿液中MBP水平与不孕患者受精率、优质胚胎率及囊胚形成率等早期助孕结局的关系。同时将40只3周龄清洁级ICR雌性小鼠简单随机抽样分为对照(玉米油)组和低、中、高剂量DBP染毒组(300 mg/kg、600 mg/kg、1200 mg/kg)四组,每组10只。将各组雌鼠促排卵与非染毒雄鼠合笼后,取受精卵,比较各组间胚胎卵裂率、囊胚形成率和着床位点的差异。结果在控制了混杂因素后,患者尿液中MBP浓度与受精率、优质胚胎数、囊胚形成数及囊胚形成率呈负相关(P=0.01、P=0.04、P=0.03、P=0.05)。DBP低剂量组、中剂量组、高剂量组的小鼠受精后胚胎卵裂率[58.38%(115/197)、43.42%(99/228)、36.60%(71/194)]、囊胚形成率[54.78%(63/115)、44.44%(44/99)、39.44%(28/71)]及胚胎着床位点数(12.20±3.03、11.80±2.68、11.60±2.41)明显低于对照组小鼠[87.12%(142/163),P<0.001;86.62%(123/142),P<0.001;16.80±0.84,P<0.001)。DBP低剂量组卵裂率高于中剂量组、高剂量组,差异有统计学意义(P=0.002,P<0.001)。DBP低剂量组囊胚形成率高于DBP高剂量组,差异有统计学意义(P=0.042)。DBP染毒的三组小鼠之间胚胎着床位点数差异无统计学意义(P>0.05)。结论DBP暴露与不孕女性临床结局存在相关性,且DBP暴露对小鼠早期胚胎发育产生毒性损伤。

Objective To explore the influence of di-n-butyl phthalate(DBP)exposure levels on early reproductive outcomes in vivo in infertile women,and its influence on early embryonic development of DBP exposed mice.Methods This study population sampling selected 112 patients according to the tail number of the medical record number who undergwent in vitro fertilization-embryo transfer(IVF-ET)treatment for tubal factor infertility from February to October 2019 in Reproductive Medicine Center of Henan Province People's Hospital.The concentration of DBP metabolite mono-n-butyl phthalate(MBP)in patients'urine was determined by HPLC tandem mass spectrometry.Multiple regression model analysis was used to analyze the relationship between urine level of MBP and early reproductive outcomes in patients with infertility.At the same time,403-week-old clean grade ICR infected female mice were randomly divided into control(corn oil)group and low-,middle-and high-dose DBP infected(300 mg/kg,600 mg/kg,1200 mg/kg)groups,with 10 mice in each group.Fertilized eggs were collected from female ovulation induction and non-infected male mice in each group,and the differences of embryo cleavage rate,blastocyst formation rate and landing point were compared.Results After controlling for the mixed factors,the concentration of MBP was negatively correlated with the fertilization rate,the number of good-quality embryos,the number of blastocyst formation and the rate of blastocyst formation as the urine level increased(P=0.01,P=0.04,P=0.03,P=0.05).There were statistically significant differences in cleavage rate between the low-dose group and the middle-dose group[58.38%(115/197)vs.43.42%(99/228),P=0.002],between the low-dose group and the high-dose group[58.38%(115/197)vs.36.60%(71/194),P<0.001],and no statistically significant difference was observed between the middle-dose group and the high-dose group(P>0.05).There was a statistical difference in blastocyst formation rate between the low-dose group and the high-dose group[54.78%(63/115)vs.39.44%(28/71),P=0.042],and no statistical difference was observed between the other two groups(P>0.05).There was no statistically significant difference in implantation sites among the three groups of mice exposed to DBP(P>0.05).Conclusion There was a correlation between DBP exposure and clinical outcomes in infertile women,and DBP exposure caused toxic damage to early embryonic development in mice.