精子透明质酸结合试验阳性率与诱发顶体反应率预测精子形态异常对受精的影响

Sperm-hyaluronan binding assay rate and induced acrosome reaction rate to predict the influence of abnormal sperm morphology on fertilization

目的探讨精子形态异常对受精影响的原因,为畸形精子症患者受精方式的选择提供参考。方法回顾性队列研究分析2016年1月至2020年3月期间于深圳市人民医院生殖医学中心首次进行常规体外受精(in vitro fertilization,IVF)的助孕周期,根据精子正常形态率(normal sperm morphology rate,NSMR)分为4组,A组:IVF精子形态正常(NSMR≥4%,750个周期),B组:IVF轻度畸形精子症(2%≤NSMR<4%,277个周期),C组:IVF中度畸形精子症(1%≤NSMR<2%,110个周期),D组:IVF重度畸形精子症(0%≤NSMR<1%,49个周期),比较各组的正常受精率、受精失败率(受精率<30%)、完全受精失败率(受精率=0)以及受精功能相关指标:2 h酪氨酸磷酸化率、透明质酸结合试验(hyaluronan-binding assay,HBA)阳性率、顶体酶含量、自发顶体反应率、诱发顶体反应率。结果①D组IVF正常受精率[52.4%(18.3%,69.0%)]显著低于A组[60.0%(45.5%,75.0%),P=0.008]和B组[60.0%(42.9%,75.0%),P=0.028];IVF受精失败率[22.4%(11/49)]显著高于A组[5.5%(41/750),P<0.001]和B组[8.3%(23/277),P=0.018];IVF完全受精失败率[14.3%(7/49)]显著高于A组[2.7%(20/750),P=0.006]。多因素logistics回归分析也显示D组IVF正常受精率显著低于A组(OR=0.433,P=0.008),受精失败风险(OR=5.426,P<0.001)、完全受精失败风险(OR=8.194,P<0.001)显著高于A组。②B、C、D组HBA阳性率[75.0%(62.3%,83.0%),71.0%(58.0%,81.0%),68.0%(48.0%,76.5%)]均显著低于A组[80.0%(71.0%,85.0%),均P<0.001];C、D组诱发顶体反应率[32.3%(26.5%,40.8%),28.8%(24.2%,43.0%)]均显著低于A组[37.8%(30.5%,46.8%),P<0.001,P=0.009]。③Spearman相关分析结果显示精子正常形态率与HBA阳性率(r=0.259,P<0.001)和诱发顶体反应率(r=0.202,P<0.001)正相关。④以精子HBA阳性率、诱发顶体反应率、NSMR为自变量,对NSMR<4%的IVF周期受精率(IVF受精率<30%)的受试者工作特征(receiver operator characteristic,ROC)曲线分析,HBA阳性率的截断值是73.5%,敏感度为51.4%,特异度为73.8%,曲线下面积(area under curve,AUC)(95%CI)=0.643(0.559~0.726),P=0.002;诱发顶体反应率的截断值是28.9%,敏感度为72.1%,特异度为50%,AUC(95%CI)=0.599(0.497~0.700),P=0.036;正常形态率的截断值是1.45,敏感度为77.8%,特异度为42.9%,AUC(95%CI)=0.605(0.509~0.701),P=0.025。结论畸形精子可能通过影响受精功能指标HBA阳性率、诱发顶体反应率从而影响IVF受精,建议对于畸形精子症患者,尤其重度畸形精子症(0%≤NSMR<1%)患者,进入促排卵周期后,男方行精子受精功能检测,包括HBA阳性率、诱发顶体反应率,如果取卵当日处理后精液符合常规IVF受精的要求,但前期受精功能检测HBA阳性率<73.5%,诱发顶体反应率<28.9%,建议行短时受精观察、卵胞质内单精子注射(intracytoplasmic sperm injection,ICSI)或half-ICSI,以降低IVF受精失败的风险。

Objective To explore the cause of abnormal morphology sperm on fertilization from insemination function and provide reference for the selection of fertilization methods for teratozoospermia patients.Methods Through a retrospective cohort study of their first in vitro fertilization(IVF)treatment cycles in Reproductive Medicine Center of Shenzhen People's Hospital from January 2016 to March 2020,all patients were divided into four groups according to the normal sperm morphology rate(NSMR),group A:IVF normal sperm morphology(NSMR≥4%,n=750),group B:mild teratozoospermia(2%≤NSMR<4%,n=277),group C:moderate teratozoospermia(1%≤NSMR<2%,n=110),group D:severe teratozoospermia(0%≤NSMR<1%,n=49).We compared normal fertilization rate,fertilization failure rate(fertilization rate<30%)and total fertilization failure rate(fertilization rate=0)among the four groups and we also compared insemination function indexs:2 h tyrosine phosphorylation rate,hyaluronan-binding assay(HBA)positive rate,content of acrosin,spontaneous acrosome reaction rate and induced acrosome reaction rate.Results 1)The normal fertilization rate of group D[52.4%(18.3%,69.0%)]was significantly lower than that of group A[60.0%(45.5%,75.0%),P=0.008]and group B[60.0%(42.9%,75.0%),P=0.028];the fertilization failure rate[22.4%(11/49)]was significantly higher than that of group A[5.5%(41/750),P<0.001]and group B[8.3%(23/277),P=0.018];the total fertilization failure rate[14.3%(7/49)]was significantly higher than that of group A[2.7%(20/750),P=0.006].Multivariate logistic regression models:the normal fertilization rate of group D was significantly lower than that of group A(OR=0.433,P=0.008),and the risk of fertilization failure(OR=5.426,P<0.001)and total fertilization failure(OR=8.194,P<0.001)were significantly higher than those of group A.2)HBA positive rate in groups B,C,D[75.0%(62.3%,83.0%),71.0%(58.0%,81.0%),68.0%(48.0%,76.5%)]was significantly lower than that in group A[80.0%(71.0%,85.0%),all P<0.001]and induced acrosome reaction rate in group C and group D[32.3%(26.5%,40.8%),28.8%(24.2%,43.0%)]was significantly lower than that in group A[37.8%(30.5%,46.8%),P<0.001,P=0.009].3)Spearman correlation analysis showed that spem normal morphology rate was positively correlated with HBA positive rate(r=0.259,P<0.001)and induced acrosome reaction rate(r=0.202,P<0.001).4)Receiver operating characteristic(ROC)curve analysis was performed to determine a cut-off value using HBA positive rate,induced acrosome reaction rate and sperm normal morphology rate as independent variables with the fertilization rate of IVF cycles(normal sperm morphology rate<4%)dichotomized at 30%.The best cut-off value of HBA positive rate obtained was 73.5%with a sensitivity of 51.4%and specificity of 73.8%[area under curve(AUC)(95%CI)=0.643(0.559-0.726),P=0.002];the cut-off value of induced acrosome reaction rate was 28.9%with a sensitivity of 72.1%and specificity of 50%[AUC(95%CI)=0.599(0.497-0.700),P=0.036];the cut-off value of normal sperm morphology rate was 1.45%with a sensitivity of 77.8%and specificity of 42.9%[AUC(95%CI)=0.605(0.509-0.701),P=0.025].Conclusion Abnormal morphology sperm may affect IVF fertilization by HBA positive rate and induced acrosome reaction rate.For teratozoospermia patients,especially for the severe teratozoospermia(0%≤NSMR<1%),we recommend that HBA positive rate and induced acrosome reaction rate are tested after ovulation induction treatment.If the post-treatment sperm meets the requirements of routine IVF fertilization on the day of retrieved oocytes,but HBA positive rate<73.5%,induced acrosome reaction rate<28.9%,short time IVF or intracytoplasmic sperm injection(ICSI)or half-ICSI is recommended to minimize IVF fertilization failure.