精子功能指标在辅助生殖助孕方式选择中的作用

目的评估精子功能参数对精子常规体外受精能力的预测价值,探寻有效选择受精方式的新指标。方法回顾性分析2017年1月至2019年12月于我院首次行体外受精-胚胎移植(IVF-ET)短时受精的429个周期的临床资料,根据受精结局不同分为2组:受精失败或受精率<30%的患者为早补救卵胞浆内单精子注射(R-ICSI)组(n=98);受精率≥30%的为短时IVF组(n=331)。比较两组患者精子前向运动(PR)、精子浓度、精子正常形态率、顶体酶活性、顶体反应(AR)、酪氨酸磷酸化(TP)及透明质酸结合试验(HBA)等指标;采用Logistic回归分析影响受精的独立危险因素,并绘制受试者工作曲线(ROC曲线)评估各项危险因素对IVF受精率的预测价值。结果两组患者精子的正常形态率、顶体酶活性、自发AR和TP比较均无显著性差异(P>0.05);R-ICSI组患者的精子浓度、PR、诱发顶体反应(IAR)和HBA均显著低于短时IVF组(P<0.05)。Logistic回归分析显示,精子PR、IAR和HBA为IVF受精率的独立危险因素;ROC曲线显示精子PR、IAR、HBA和联合预测新变量(Pre-1)的曲线下面积(AUC)分别为0.66、0.62、0.64和0.70。结论精子功能指标中IAR和HBA是影响IVF受精率的独立危险因素,其对辅助生殖助孕方式的选择有一定指导意义。     

Acrosin activity in patients with idiopathic infertility

Acrosin is a sperm acrosomal enzyme that is involved in the acrosome reaction, the binding of spermatozoa to the zona pellucida, and fertilization. This study was designed to determine whether sperm acrosin measurements can identify subpopulations of infertile or subfertile patients that are not recognized by routine semen analyses. We measured the total acrosin activity of ejaculates in a group of 19 men (15 suspected subfertile patients and 4 fertile donors). The acrosin activity was measured in liquefied semen specimens using the methodology described by Kennedy et al. [1989) J Urol 10:221-231). Ten patients in the suspected subfertile group had a mean acrosin value of 7.8 microIU acrosin/million sperm, which is clearly in the abnormal range (less than 14 microIU/10(6) sperm). Three patients had a mean acrosin value of 20.1 microIU/10(6) sperm, which is in the indeterminate range. Two other patients and four proven fertile donors had acrosin values in the normal range (greater than 25 microIU/10(6) sperm) (Agarwal A, Loughlin KR (1990): 2d International Meeting of Andrology, Como, Italy; Abstr 22). The normal fertile controls had a mean acrosin value of 32.5 microIU/10(6) sperm.     

精子形态率、顶体酶活性对IVF受精失败的影响

目的 探讨精液中精子形态率及顶体酶活性与IVF受精失败的关系.方法 回顾性分析2014年在本中心行IVF新鲜周期的982个周期患者,根据精子形态分析结果,将正常精子形态百分率(x)分为5组,包括:1组:x≥4％,2组:x＜1％,3组:1％≤x＜2％,4组:2％≤x＜3％,5组:3％≤x＜4％.根据顶体酶活性分为正常组及异常组.结果 IVF受精失败组(受精率＜30％)与正常组比较,正常精子形态率及顶体酶活性差异有统计学意义(P＜0.01).精子形态率比较,2、3、4组与1组比较,差异有统计学意义(P＜0.01).5组与1组比较,差异无明显统计学意义(P＞0.05).顶体酶活性正常组与异常组比较,受精失败率差异有统计学意义(P＜0.01).结论 精子形态率及顶体酶活性异常可导致IVF受精失败率升高.     

New method of evaluating sperm morphology with predictive value for human in vitro fertilization

A prospective study was planned to evaluate sperm morphology as a parameter to predict the fertilization outcome in an in vitro fertilization program. Couples applying to in vitro fertilization were admitted into this project when the sperm concentration was greater than 20 million per mL and motility greater than 30 per cent. Based on new strict criteria for evaluating normal sperm morphology, patients were divided prospectively into 2 groups. In group I (25 patients) normal sperm morphology was less than 14 per cent, and in group II (71 patients) normal sperm morphology was greater than 14 per cent, using a threshold established previously. Multiple regression analysis was used to evaluate different parameters: concentration, motility, and morphology against the dependent variables, fertilization, and cleavage. The only factor which was significantly correlated with fertilization and cleavage was normal sperm morphology (P less than 0.0001). The fertilization rate (per oocyte) and the cleavage rate were 49.4 per cent and 47.6 per cent in group I and 88.3 per cent and 87 per cent in group II (P less than 0.0001). The ongoing pregnancy rate per laparoscopy and per embryo transfer was 4 per cent and 5.5 per cent, respectively, in group I and 18.3 per cent and 18.5 per cent, respectively, in group II (no significant difference). This study demonstrates the value of analyzing sperm morphology using the criteria recommended in terms of predicting fertilization and perhaps pregnancy outcome. Patients can be better counseled and the probability of fertilization or no fertilization can be more accurately established. Furthermore a trend is shown in the pregnancy rate that may indicate the importance of the male genome in establishing a pregnancy.     

异常形态精子对植入前胚胎发育和妊娠结局的影响

目的探讨异常形态精子(畸形率≥98%)对植入前胚胎发育及妊娠结局的影响。方法采用回顾性队列研究,分析2017年1~12月在唐都医院妇产科生殖医学中心行ART助孕的2419例患者临床资料,根据异常形态精子分为3组,即IVF对照组(畸形率≤96%,n=2129)、IVF实验组(畸形率≥98%,n=90)和ICSI实验组(畸形率≥98%,n=200)。比较3组间植入前受精失败率(受精率<30%)、正常受精率、可用胚胎率等胚胎发育参数和着床率、临床妊娠率、流产率及活产率等妊娠结局的差异。结果(1)胚胎发育结果:组间比较,IVF实验组受精失败率显著高于IVF对照组(P<0.05),ICSI实验组的受精失败率为0;ICSI实验组正常受精率显著高于IVF对照组和IVF实验组(P<0.05);IVF实验组可用胚胎率显著低于IVF对照组和ICSI实验组(P<0.05)。(2)妊娠结局:单因素分析结果显示,与IVF对照组、ICSI实验组相比,IVF实验组的着床率、临床妊娠率、流产发生率和活产率差异均有统计学意义(P<0.05);IVF对照组和ICSI实验组组间妊娠结局指标比较均无显著性差异(P>0.05)。(3)Logistic多因素分析显示:IVF实验组的受精失败风险显著高于IVF对照组(P=0.002),可用胚胎率、活产率显著低于IVF对照组(P=0.002);ICSI实验组的正常受精率显著高于IVF对照组(P=0.05)。结论对于活力正常、但异常形态精子率≥98%的患者,采用ICSI授精方式,能降低受精失败风险,提高正常受精率和可用胚胎率,同时提高妊娠率和活产率并降低流产发生率。     

Association between sperm cell chromatin condensation, morphology based on strict criteria, and fertilization, cleavage and pregnancy rates in an IVF program

Summary. In this study, a total of 95 ejaculates from infertile patients were investigated morphologically according to Kruger's strict criteria and 78 of the 95 ejaculates were stained for chromatin condensation with acidic aniline blue. Patients were divided into two groups based on the percentage of morphologically normal spermatozoa as follows: Men with normal sperm morphology <14% (Group I), and men with normal morphology >14% (Group 2). The relationship between percentage of normal sperm morphology and fertilization, cleavage and pregnancy rate was analysed. The rates were 33.7%, 57.1% and 0.0% respectively, in the first group. The corresponding values for the second group were 76.1%, 68.2% and 32.1%. The fertilization and pregnancy rates correlate significantly with morphologically normal spermatozoa.
In regard to the percentage of morphologically normal spermatozoa stained with aniline blue, patients were divided into two groups: patients with 0–20% stained spermatozoa (Group I) and those with >20% (Group 2). Fertilization and pregnancy rates were higher in the first group than in the second group (79.9%, 52.8% vs. 58.8%, 29.5%).
The results demonstrate that chromatin condensation visualized by aniline blue staining is a good predictor for IVF outcome and should be considered besides morphology by sperm assessment for patients undergoing IVF treatment.—     

人精浆抗精子抗体与顶体酶活性关系的探讨

目的 :研究精浆抗精子抗体 (AsAb)对精子顶体酶活力的影响。　方法 :用间接血凝法测定 34 32例不育男性和 6 5例生育男性精浆的AsAb ,并对其中的 2 882例不育男性精子用比色法进行了顶体酶活性的测定。　结果 :34 32例不育者精浆AsAb阳性率为 10 .2 0 %,2 882例进行了顶体酶活性测定的不育者精浆AsAb阳性率为 9.37%,对照组精子顶体酶活性明显高于各不育组 (P <0 .0 0 1)。不育组AsAb阳性组与AsAb阴性组比较顶体酶活性没有明显差异 (P >0 .0 5 ) ,顶体酶活性正常与异常组AsAb阳性率比较差异无显著性 (P >0 .0 5 )。　结论 :精浆AsAb对精子顶体酶活性没有影响。     

Relationship of acrosin activity to sperm function tests

Summary.  Acrosin activity of spermatozoa from infertile patients and fertile volunteers was measured. Acrosin activity of spermatozoa from asthenozoospermic patients and patients with unexplained infertility was lower than that from fertile volunteers. We utilize the zona-free hamster egg sperm penetration test to select candidates for conventional in vitro fertilization among unexplained infertile patients. The zona-free hamster egg sperm penetration test, however, requires several hours and special equipment which are not used in the clinical setting. It is preferable that other sperm function tests or a combination of them can replace this test. Thus three distinct tests of sperm function, namely, acrosin activity, Penetrak® test, hypo-osmotic swelling test, were compared with the hamster test, using spermatozoa from patients with unexplained infertility.
A combination of the Penetrak® test and measurement of acrosin activity could predict the results of the zona-free hamster egg sperm penetration test with 88.2% accuracy. Thus, the hamster test should be done when either Penetrak® test or measurement of acrosin activity showed abnormal values.     

优选前后精子顶体酶活性与IVF受精率相关性的研究

目的探讨优选处理前和处理后精子顶体酶活性的变化,及与体外受精(IVF)受精率的相关性。方法采用分光光度比色法,对接受IVF治疗的53例不育夫妇男方精液,分别测定优选处理前和处理后精子顶体酶活性,分析其与IVF受精率的相关性。结果优选处理后精子顶体酶活性与优选前比较有显著性差异(P<0.05);达到常规IVF标准并选择常规IVF治疗者,优选后的精子顶体酶活性与IVF受精率有相关性,精子顶体酶活性降低与IVF受精率降低有关。结论精子顶体酶活性与IVF受精率有相关性,并且通过精子顶体酶活性可以预测IVF受精率。     

低氧对大鼠精子体外顶体反应和顶体酶、透明质酸酶活性的影响

目的研究低氧对大鼠精子顶体酶、透明质酸酶活性的影响以及对精子体外顶体反应发生的影响。方法成年Wistar大鼠随机分为4个组:常氧对照组、低氧5d组、低氧15d组和低氧30d组。低氧组置低压舱内模拟高原5000m,分别低氧5、15和30d。采用明胶薄膜法、透明质酸底物转化法、溶血磷脂胆碱(LPC)诱导金霉素荧光染色法,测定低氧对附睾尾精子顶体酶、透明质酸酶活性和体外顶体反应发生率的影响。结果低氧5、15和30d组大鼠精子顶体反应发生率,与常氧对照组相比均显著降低,分别由常氧对照组的(30.5±3.5)%,下降至(11.7±0.9)%(P<0.01)、(10.8±1.0)%(P<0.01)和(10.0±1.4)%(P<0.01);低氧5、15和30d组大鼠精子顶体酶阳性率降低,分别由常氧对照组的(81.67±7.16)%,下降至(54.17±3.82)%(P<0.01)、(30.00±3.92)%(P<0.01)和(43.00±3.63)%(P<0.01);低氧各组透明质酸酶活性无显著改变(P>0.05)。结论低氧抑制了精子顶体酶活性和顶体反应发生。     

Assessment of sperm functions in infertile patients with varicoceles

Acrosin activity, acrosome reaction and nuclear chromatin condensation were studied in 24 infertile patients with varicoceles and 26 fertile men with or without varicocele. Chromatin condensation, assessed by aniline blue staining, and acrosin activity, evaluated by gelatinolysis technique, were significantly affected in the group of infertile patients. Defective chromatin condensation and defective acrosin activity were detected in 67% and 50% of the infertile patients, respectively. No significant difference was found between the two groups regarding the acrosome reaction, which was assessed by the triple staining technique after exposure of spermatozoa to low temperature (4 degrees C). This study identified a subgroup of infertile patients with normal standard semen parameters but impaired sperm functions. Results of the sperm function tests and standard semen parameters were not correlated. Therefore, it is concluded from this study that important sperm functions are impaired in patients with varicocele and that the gelatinolysis technique and aniline blue staining are effective tools for assessment of the fertilization potential of varicocele patients.     

Defective sperm decondensation: a cause for fertilization failure

The study aimed to evaluate the role of chromatin packaging (CMA3 staining), sperm morphology during sperm-zona binding, sperm decondensation and the presence of polar bodies in oocytes that failed in vitro fertilization (IVF). The percentage CMA3 staining categorized the data into three groups, < 44%, n = 10; > or = 44-59%, n = 10; and > or = 60%, n = 29. Morphology groups were < or = 4% (n = 11); > 4-14% (n = 19); and > 4% (n = 19). One hundred and seventy-two oocytes that failed IVF were evaluated for sperm-zona binding, ooplasma penetration and sperm decondensation. Odds ratio analyses indicated that being in the > or = 60% CMA3 staining group resulted in a 15.6 fold increase in the risk of decondensation failure, relative to CMA3, staining of < 44%. For morphology, there was a 2.17 fold decrease in the risk of fertilization failure in the morphology group with > 4-14% normal cells, while it increased 2.45 fold for the morphology group with < or = 4% normal cells. Using CMA3 fluorescence to discriminate, 51% of the oocytes in the group with elevated CMA3 fluorescence had no sperm in the ooplasma compared to 32% and 16% penetration failure in the CMA3 staining groups > or = 44-59% and < 44%, respectively. Sperm chromatin packaging quality and sperm morphology assessments are useful clinical indicators of human fertilization failure. Immunofluorescence techniques could be used to provide a clear diagnosis of failed fertilization.     

Usefulness of sperm penetration assay in fertility predictions

The competence of the sperm penetration assay (SPA) to predict male fertility, as determined by normal sperm morphology and the fertilizing potential, as shown by human in vitro fertilization (IVF), was investigated. A significant correlation was obtained between normal sperm morphology and the SPA (phi = 0.623). A weaker correlation was however obtained with human IVF (phi = 0.397). Notwithstanding this weak association, a positive SPA (greater than 10%) was highly predictive (95%) of human IVF success. In contrast, a negative SPA (less than or equal to 10%) was associated with a high rate of false-negatives (65%). The SPA does however warn that a male factor may be present, as the mean fertilization rate of this group of patients was markedly reduced. The preincubation period for the spermatozoa did not play a major role in the predictive ability of a SPA outcome.     

Critical assessment of sperm morphology before and after double wash swim-up preparation for in vitro fertilization

Critical assessment of sperm morphology using specific and stringent criteria is predictive of the subsequent ability of those sperm to fertilize oocytes in in vitro fertilization (IVF). Previous studies have evaluated sperm morphology prior to sperm preparation and, thus, have not assessed the actual sperm used for insemination. We studied the impact of a double wash swim-up technique used for IVF on sperm morphology using the strict criteria of Kruger et al. [5, 6] in 73 consecutive patients undergoing IVF. Pre- and postswim-up morphological assessments were done in a prospective, randomized, and blinded fashion. The mean percentage of normal forms pre- and postswim-up was 19.8% and 23.4%, respectively, an improvement of 18% (p less than 0.05) with 62 of 73 patients showing improvement. Significantly, analysis of the 27 patients with abnormal morphology on initial assessment (normal forms less than or equal to 14%) revealed an improvement in percentage of normal forms from 9.0% to 21.5%, a mean increase of 239% (p less than 0.005). Eighteen of these 27 patients showed improvement in their percentage of normal forms, eight were unchanged, and only one patient had a decline in their percentage of normal forms. We conclude that the double wash swim-up preparation used for IVF substantially improves the percentage of sperm with normal morphology, and that the benefit is most substantial in those cases where the percentage of normal morphology is abnormal.     

217例男性不育症精子顶体酶检测与分析

目的　研究人精子顶体酶与男性不育之间的关系。　方法　 2 1 7例不育男性 ,年龄 2 4～ 43岁 ,根据精液常规检查结果分为不育 A、B两组 ;46例生育男性为对照组。手淫留取精液标本 ,常规洗涤后孵育 ,采用固定明胶底膜法检测精子顶体酶。　结果　正常生育组的精子顶体酶活性阳性率为 86.5% ,酶活性亮区直径为 42 .9μm。不育 A组酶活性阳性率为 57.0 % ,酶活性亮区直径为 3 8.5μm。不育 B组酶活性阳性率为3 5.2 % ,酶活性亮区直径为 2 7.5μm。经统计学处理 ,生育组与不育组之间精子顶体酶阳性率差异有显著性 ( P<0 .0 1 )。　结论　顶体酶阳性率和活力低下是男性不育的一个重要原因 ,精子顶体酶是受精过程中的关键酶     

Relation of human sperm acrosin activity and fertilization in vitro

The aim of the study described here was to determine the possible contribution of the acrosin activity test to routine semen analysis in enhancing the precision of the prognosis of IVF success in a group of patients in which the contribution of the egg factor to infertility was ruled out (20 cases) compared to a control IVF group (39 cases). Semen analysis, acrosin activity and acrosome ultrastructure were determined for all semen samples. The group with high fertilization rates was comprised of normozoospermic patients while the group with low fertilization rates was comprised of astheno-teratozoospermic patients. The mean acrosin level of the positive IVF group was significantly higher than that of the negative group (51.7 ± 33.2 and 28.6 ±13.7, respectively). Two parameters: per cent motile spermatozoa and acrosin level, were found to have a significant positive correlation with subsequent successful IVF ( r = 0.36, P < 0.006; r = 0.37, P < 0.004, respectively); and agenesis of the acrosome was found to have a significant negative correlation ( r = -0.33, P < 0.01). The ability of these parameters to correctly predict fertilization success was 59%, with 5% false positive, among which 15.4% was predicted solely by the acrosin level (above 54 μIU 10⁶ cells⁻¹) and 23% solely by per cent motile spermatozoa (above 50%). Abnormalities of the acrosome ultrastructure did not contribute further to the correct classification. The apparent clinical benefit of the acrosin level test is discussed.     

Influence of strict sperm morphology on the results of classic in vitro fertilization

Objective: To determine the influence of the concentration of oval spermatozoa according to the strict morphology criterion in men with normal sperm concentration following the World Health Organization criteria on the results of classic IVF. Materials and Methods: Based on review of patient charts, this study included infertile couples presenting with female causes for infertility or unexplained infertility, in whom men presented with normal spermogram values for sperm concentration, sperm motility, volume of ejaculate and total sperm count after semen processing greater than 20 million. Based on the value obtained in strict sperm morphology, patients were divided into three groups: in Group A, patients with values between 0 % and 4 %; in group B, between 5 % and 14 %, and in group C, patients with sperm morphology greater than 14 %. The outcomes analyzed were oocyte fertilization rate, biochemical pregnancy rate, clinical pregnancy rate and rate of liveborns. Results: A total of 244 cases met the inclusion criteria, 27 of them in group A, 165 in group B, and 52 in group C. The mean fertilization rate and the rate of liveborns were, respectively: 71.9 % and 33.3 % in group A; 80.9 % and 24.2 % in group B, and 78.8 % and 28.8 % in group C. There was no statistical difference among the groups in any of the outcomes analyzed. Conclusion: The values of strict sperm morphology, as proposed by Kruger and adopted by the World Health Organization, had no infl uence on the results of classic in vitro fertilization in the studied sample.     

精子畸形率对体外受精-胚胎移植治疗结局的影响

目的回顾性分析我中心接受常规体外受精（IVF）及卵胞浆内单精子注射（ICSI）治疗中,男方精子畸形率对受精率、胚胎质量及临床结局的影响。方法选取本中心2008年9月至2010年5月接受IVF的344对及ICSI的178对夫妇,分为常规IVF组和ICSI组,组内按照男方精子畸形率分为正常形态组（IVF266／ICSI76）和畸精子症组（IVF78／ICSI102）。受精后分别统计IVF及ICSI两组内畸精子症组和正常形态组正常受精率、优质胚胎率、种植率、临床妊娠率及流产率的差别。结果在IVF中,畸精子症组和正常形态组的正常受精率、种植率、临床妊娠率及流产率分别为64．32％／60．09％、33．78％／37．02％、42．03％／54．62％及12．5％／4．23％。两组间受精率无显著性差别,畸精子症组的临床妊娠率显著性低于正常形态组,而早期流产率显著高于正常形态组（P〈0．05）;ICSI组中,畸精子症组和正常形态组的正常受精率、种植率、临床妊娠率及流产率分别为68．01％／64．59％、32．26％／33．78％、43．75％／52．63％及4．76％／5％。畸精子症患者的临床妊娠率较正常组显著性降低（P〈0．05）。将两种受精方式的畸精子症组间比较,IVF的患者早孕流产率显著高于ICSI者（P〈0.05）。结论常规IVF中畸精子症不影响正常受精。对于畸精症子患者,其临床妊娠率均较精子形态正常组低,但是采用ICSI治疗可以显著降低早孕流产率。‘     

精子畸形程度对常规体外受精-胚胎移植治疗的影响

目的 探讨精子畸形程度对常规体外受精(IVF)-胚胎移植治疗的影响. 方法 2007-2008年接受常规体外受精-胚胎移植105对夫妇男方精液进行分析,按精子正常形态百分率分为3组,正常精子形态组(精子正常形态≥15%)54例、轻度畸形精子组(精子正常形态10%～15%)26例、中度畸形精子组(精子正常形态5%～10%)25例,观察精子畸形程度对IVF受精率、卵裂率、优质胚胎率、着床率、妊娠率及流产率的影响. 结果①精子形态正常组与轻度畸形组受精率(79.4%与78.3%)、卵裂率(104.6%与98.6%)、优质胚胎率(58.1%与53.9%)、植入率(31.7%与30.8%)、妊娠率(48.1%与42.3%)及流产率(13.0%与18.2%)比较差异均无统计学意义(P>0.05).②精子形态正常组与中度畸形组受精率(63.9%)、优质胚胎率(48.2%)、植入率(16.7%)、妊娠率(24.0%)比较差异有统计学意义(P<0.05).而卵裂率(102.9%)、流产率(28.6%)比较差异无统计学意义(P>0.05).③轻度畸形组与中度畸形组受精率、优质胚胎率比较差异有统计学意义(P<0.05),植入率(30.8%与16.7%)、妊娠率(42.3%与24.0%)、卵裂率(98.6%与102.9%)、流产率(18.2%与28.6%)比较差异无统计学意义(P>0.05).结论 中度畸形精子患者常规IVF受精率、优质胚胎率、植入率和妊娠率有一定影响;轻度畸形精子对常规IVF影响不大.     

精子CMA3阳性率与IVF受精率的相关性研究

目的探讨精子色素酶A3(CMA3)阳性率与IVF受精率之间的相关性。方法收集2015年4月至2016年7月因单纯输卵管性不孕在我院生殖医学科行常规IVF助孕的156个周期,以受精率<25%为低受精,根据受精率不同分为正常受精组(134个周期)和低受精组(22个周期)。取卵日,将授精后剩余的洗涤精液行精子浓度和活动率分析,并将精子进行CMA3染色。比较两组优选后的精子参数,分析IVF受精率与精子参数及CMA3阳性率的关系。结果与正常受精组比较,低受精组精子CMA3阳性率显著升高[(20.0±4.2)%vs.(30.7±2.3)%],优选后前向运动精子的百分率显著降低[(90.4±4.8)%vs.(74.3±3.4)%](P<0.05);两组精子浓度比较无显著性差异(P>0.05)。IVF受精率与优选后的前向运动精子百分率呈正相关(r=0.76,P<0.01)。精子CMA3阳性率与优选后前向运动精子百分率(r=-0.82,P<0.01)及IVF受精率(r=-0.83,P<0.01)呈显著负相关,与精子浓度则无显著相关性(P>0.05)。结论精子CMA3阳性率与IVF受精率负相关,但其具体机制尚需进一步研究探讨。