The human acrosome reaction

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, spermzona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization (IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one woman affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pellucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morphology. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion of acrosome intact sperm in the insemination medium was related to the IVF rate. Inducing the acrosome reaction with a calcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented spermzona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding. Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normal sperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrated the zona. In contrast, fertilization and pregnancy rates were high with intracytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signal transduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler tests and treatments for the patients and also provide new leads for contraceptive development.     

Effect of human sperm chromatin anomalies on fertilization outcome post-ICSI

The aim of this study was to evaluate the effect of sperm chromatin anomalies on fertilization outcome post-intracytoplasmic sperm injection (ICSI). Therefore, along with semen parameters, Chromomycin A3 (CMA3) staining for protamine deficiency, aniline blue staining for excessive histones, SDS for sperm chromatin stability and SDS + EDTA for the ability of sperm to undergo decondensation were carried out on 55 semen samples from patients referred to the Isfahan Fertility and Infertility Center for ICSI. The results showed that among the aforementioned tests and semen parameters only CMA3 showed a significant correlation with fertilization outcome post-ICSI. Patients were also grouped according to CMA3 level of <30% or >30% or fertilization rate of <50% or >50%. The results show that the mean percentage fertilization and mean percentage of CMA3 positivity is different in both groups, respectively. The area under receiver operator characteristics curve shows that CMA3 is a highly sensitive and specific test for prediction of fertilization outcome post-ICSI. In conclusion, that sperm protamine deficiency has profound effect on fertilization failure in ICSI.     

Comparison of sibling oocyte outcomes after intracytoplasmic sperm injection and in vitro fertilization in severe teratozoospermic patients in the first cycle

To follow up the outcome of sibling oocytes subjected to both conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in the first cycles of severe teratozoospermic patients with normal sperm morphology (NSM)or=6 cell embryos on day 3 and blastocyst formation on days 5 and 6, did not differ significantly between the two groups. There was a trend towards a high pregnancy rate cycle in mixed ICSI/IVF embryo transfer (ET) (49.1%). In conclusion, this study showed that in couples with only severe teratozoospermia, there was a benefit in subjecting sibling oocytes to both IVF and ICSI in the first cycle because 24 (28.2%) cycles of total fertilization failure were avoided. Furthermore, despite initially significant higher fertilization rates in ICSI than IVF oocytes, subsequent rates of development from >or=6 cells up to blastocyst stage were the same suggesting that ICSI should be used with caution, as after day 3, ICSI-derived embryo development was compromised compared with IVF.     

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。     

Relationship between protamine deficiency with fertilization rate and incidence of sperm premature chromosomal condensation post-ICSI

After aneuploidy, sperm premature chromatin condensation (PCC) is the next prevalent cause of fertilization failure. Therefore the aim of this study was to evaluate the effect of sperm protamine deficiency on sperm PCC formation post-ICSI. Chromatin analysis was carried out on failed fertilized oocytes post-ICSI and incidences of sperm PCC were evaluated and the results were compared with the extent of protamine deficiency assessed by chromomycin A3. The results show that incidence of sperm PCC was significantly different in failed fertilized oocytes injected from semen samples with greater or less than 30% CMA3 positivity (P = 0.04). However, except for fertilization rate (P < 0.001), the mean number of MII, MI and germinal vesicles oocytes and percentage normal sperm were not significantly different between the two groups (P > 0.05). A significant correlation was observed between sperm protamine deficiency with fertilization rate. Hence sperm protamine deficiency affects fertilization rate and possibly prones sperm to PCC post-ICSI.     

IVF过程受精失败的预测

在发明和常规应用卵胞浆内单精子注射（ICSI）之前,十多年的临床体外受精（IVF）治疗实践中,受精率低下很常见,大约有20％～35％的IVF患者受精率很低（〈35％的卵子受精）和受精完全失败（所有卵子都不受精）。虽然受精失败与精子或卵子的质量有关,但相当一部分患者受精失败与精液质量或精子功能低下有很密切相关性。最常见的是严重的少精,弱精和畸形精子症患者。     

精子功能检测与男性不育诊治的新进展

传统的精液常规分析是用于判断男性生育力的最基本临床指标,但是,只依靠精液分析的结果来预测男性生育状况仍是很不准确的。精子功能正常与否,对临床选择IVF还是ICSI治疗不育症极为重要。因为IVF需要功能完全正常的精子才能受精,而ICSI的受精只需要精子的正常核DNA,不需要其它的精子功能。在发明ICSI以前,患者IVF受精失败或低下(<30%)发生率很高(20%~35%)。研究证明,这些IVF受精失败的患者主要与精子功能障碍有关。常见的是少精子症,弱精子症和畸形精子症。但是有很多患者,精液分析结果仍正常。为了提高临床对精子功能测定的准确性,文献里有很多新的精子功能试验的研究报导,比如丫啶橙(AO)测定精子DNA、精子与透明带结合和穿透、顶体诱发精子顶体反应和精子与透明质酸结合试验。精子形态测定是常规精液分析中最重要的临床指标之一。但精子形态又是最难测定准确和稳定。IVF/ICSI受精失败的人卵可以用来测定精子功能。人卵透明带选择性地与正常形态和顶体完整的精子结合,透明带诱发的顶体反应与精子穿透明带的能力有很强的相关性。在不明原因的男性不育患者中,由于透明带诱发顶体反应障碍所导致的不育症占25%左右。少精子症(精子计数<2×106/ml)和严重精子形态畸形症(严格正常形态<5%)的男性不育患者,精子-透明带结合反应缺陷的发生率很高(>70%)。这类患者用IVF治疗受精率会很低,因此只能用ICSI治疗。精子与透明质酸结合试验与精子活力和形态有很强的相关性,但它不是很有用的精子功能试验。AO测定精子DNA对预测ART的受精和妊娠率的临床意义目前还没有肯定的结论,需要进一步研究。总之,在常规精液分析时,增加一些新的精子功能试验,在临床ART中对男性不育患者的诊治会有很大的帮助。     

体外受精失败MII期人卵母细胞的免疫荧光研究

目的探讨体外受精中MII期人卵母细胞受精失败的原因。方法收集体外受精后24～48h仍未受精的MII期卵母细胞,进行免疫荧光染色和碘化丙啶(PI)复染,在荧光显微镜下对其失败原因进行分类。结果卵母细胞内未见精子的在常规体外受精(IVF)周期有55.8%,显著多于卵胞浆内单精子注射(ICSI)周期中的9.7%(P<0.01);卵母细胞活化失败两者分别为14.9%和58.1%,有显著性差异(P<0.01);原核形成和(或)迁移缺陷的在两者分别为25.3%和32.3%(P>0.05);其他异常两者分别为3.9%和0.0%。结论IVF中MII期卵母细胞的受精失败主要是缺乏精子的穿透,ICSI周期中的主要原因是卵母细胞活化不完全。     

Normal sperm morphology and chromatin packaging: comparison between aniline blue and chromomycin A3 staining

The successful implementation of ICSI has provided a unique means of allowing couples suffering from severe male infertility to achieve their reproductive goals. However, despite the great therapeutic advantages of the technique, ICSI often provides solutions to clinicians in the absence of an aetiological or pathophysiological diagnosis. The development of a sequential diagnostic schedule for patients consulting for fertility disturbances would be an ideal method of approach. Since sperm morphology recorded by strict criteria has often been correlated with fertilization failure, the present study aimed to evaluate the relationship between normal morphology and chromatin staining among fertile and subfertile men. Both chromomycin A3 (CMA3) and acidic aniline blue (AAB) were employed to record chromatin packaging quality among 58 men visiting the andrology laboratory. Intra- and interassay variations were initially recorded for fertile sperm donors. The coefficients of variation (CV) for all intra- and inter-assay assessments were < 12%. Chromatin packaging was significantly and negatively correlated with normal sperm morphology, namely r = 0.40 (P = 0.001) and r = 0.33 (P = 0.001) for CMA3 and AAB, respectively. Receiver operator characteristics illustrated sensitivity and specificity values of 75% and 82% for CMA3 and 60% and 91% for AAB, respectively. Significantly different CMA3 and AAB staining was recorded among men with severe teratozoospermia (< 4% normal forms) when compared with normozoospermic men (> 14% normal forms), namely 49% vs. 29% for CMA3 and 51% vs. 26% for AAB staining, respectively. Chromatin packaging assessments should be a valuable addition to the sequential diagnostic programme in an assisted reproduction arena.     

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。     

Sperm decondensation and semen parameters: utilization of a simple staining technique for the evaluation of human sperm decondensation

Summary. The ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin on penetration of the oocyte. Knowledge of what percentage of sperm decondenses is useful, especially in patients where other functional tests and sperm quality fail to explain the reported poor in vitro fertilization (IVF) rates. The objective of this study was (1) to compare sperm decondensation induced by either SDS/EDTA or heparin with semen parameters (volume, concentration, motility and morphology), and (2) to evaluate the use of a simplified staining technique (Diff Quik^R [DQ]) in comparison with the standard phase contrast method (Rose Bengal-[RB]). Randomly selected semen samples from 31 men attending an assisted reproductive programme were analysed for basic semen parameters and decondensation with SDS/EDTA and heparin. Two staining methods for the evaluation of decondensation were compared (phase contrast microscopy after Rose Bengal [RB] staining and light microscopy after Diff Quik^R (DQ) staining). Moderate and grossly swollen sperm heads were recorded. Semen samples included both fertile and unfertile semen parameters. Sperm decondensation results showed poor to moderate correlations with semen parameters. The SDS/EDTA (DQ) (moderate forms) showed a significant negative correlation (r = -0.46) with seminal volume and and a significant positive correlation (r = 0.41) with normal sperm morphology. The heparin (DQ) (moderate forms) decondensation showed a significant positive correlation with motility (r = 0.61) and sperm concentration (r = 0.43). The DQ method was preferred over the RB method due to its optical and storage advantage. Sperm decondensation by SDS/EDTA and heparin have limited use in the IVF laboratory as they correlate poorly with semen parameters. Future studies should investigate the use of an ooplasmic factor similar to nucleoplasmin in Xenopus laevis egg.     

IVF—ET中未受精卵母细胞行早期（5hrs）补救ICSI授精后胚胎发育潜能

在常规应用卵胞浆内单精子注射（ICSI）之前,常规体外受精（IVF）受精率低下很常见,大约有20％～35％的IVF患者会发生受精率很低（〈35％的卵子受精）甚至受精完全失败（所有卵子都不受精）。卫生部辅助生殖技术管理条例规定,在应用辅助生殖技术助孕的治疗中,对以女方输卵管堵塞、排卵障碍、子宫内膜异位症或不明原因等不孕因素为主的患者,     

A retrospective comparison of pregnancy outcome following conventional oocyte insemination vs intracytoplasmic sperm injection for isolated abnormalities in sperm morphology using strict criteria

Initial in vitro fertilization (IVF)-embryo transfer studies found poor fertilization and pregnancy rates following conventional fertilization of oocytes when using sperm with <4% normal morphology using strict criteria. Some consider today that sperm with only < or =5% normal morphology using strict criteria are associated with infertility. However, other studies have disputed the diagnostic potential of low strict morphology in identifying subnormal male fertility. Based on the original studies most IVF centers perform intracytoplasmic sperm injection (ICSI) when the sperm shows low morphology using strict criteria to allow selection of normal sperm. However, ICSI adds extra time for the embryologist and extra expense for the infertile couple. The present study retrospectively compared fertilization, pregnancy, and implantation rates according to the 2 methods of oocyte fertilization with sperm having normal morphology using strict criteria of < or =5% in women < or =39 years. All fresh embryo transfers were performed on day 3. There was a significantly higher fertilization rate with ICSI. However, there were significantly higher clinical and delivered pregnancy and implantation rates following conventional insemination. The rate of canceled transfers due to no available embryo was 1.9% with conventional insemination vs 1.5% for ICSI in women with adequate egg reserve. Hopefully, this retrospective study will generate interest in a prospective study.     

ICSI as an effective therapy for male factor with antisperm antibodies

This study was conducted to evaluate if in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) is an effective treatment for infertility complicated by the presence in the male partner of sperm autoantibodies. Over a 1-year study period comparisons of fertilization, pregnancy, and implantation rates were made in couples where the male partner was negative or weakly positive for sperm autoantibodies (<50%) (gr 1); autoantibodies were strongly positive (>80%) (gr 2); or autoantibodies were moderately positive (50-80%) (gr 3). Only patients having oocytes fertilized by ICSI were included. The fertilization, clinical pregnancy, implantation, and miscarriage rate for group 1 (n = 67) was 56, 43, 21, and 14%. Comparable values for group 2 (n = 20) were 55, 40, 23, and 25%, and for group 3 (n = 6) were 63, 33, 23, and 0%. IVF with ICSI demonstrates comparable fertilization, pregnancy, implantation, and miscarriage rates in female partners of males with and without sperm autoantibodies.     

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=...     

Correlation between sperm morphology, acrosin, and fertilization in an IVF program

Acrosin, a neutral proteinase, is located within the acrosome. The aim of this study was to evaluate acrosin concentrations in patients with severe damage of the sperm head and to determine whether acrosin concentration could predict the chances of fertilization in an IVF program. Sixty patients were accepted into this study, prospectively. The patients were divided into two groups, those with a normal morphology of less than 14% (group I, n = 33) and those with normal morphology less than 14% (group II, n = 27). All patients had a sperm concentration of less than 20 million sperm/ml and less than 30% progressively motile sperm. The acrosin assays were performed on the semen sample obtained on the day of IVF. Routine IVF insemination procedures were used, and only mature oocytes were considered. The only factor that showed a significant correlation of fertilization was normal morphology (p less that 0.01). The mean acrosin level was 73.4 /+- 38.6 mED/10 million sperm in group I and 70.9 /+- 42.7 mIU/10 million sperm in group II (no significant difference). The fertilization rate in group I was 45.4% and in group II, 77.7% p less than 0.002). Acrosin levels were not significantly different in patients with and without fertilization (72.0 /+- 42.1 and 73.6 /+- mIU/10 million sperm, respectively).     

In vitro decondensation of nuclear chromatin of human spermatozoa: assessing fertilizing potential.

Spermatozoan nuclear chromatin is in a highly condensed state prior to fertilization. In vivo decondensation occurs in the ooplasm and is essential for successful fertilization and the formation of male pronucleus and the zygote to occur. The chromatin of spermatozoa and nucleus can undergo in vitro decondensation with sodium dodecyl sulfate (SDS) and 6 mM ethylene diamine tetraacetic acid (EDTA). The ability of sperm to decondense in vitro was compared with their ability to fertilize human oocytes in vitro. Spermatozoa from normal samples were studied for their decondensation ability as regards their fertilizing performance in an in vitro fertilization (IVF) program. Fertilization occurred when the decondensation percentage of sperm nuclear chromatin was more than 70%. The effective sperm count was significantly (p less than 0.05) lower in the unfertilized group. This is a new diagnostic technique to assess sperm-fertilizing potential at the initial evaluation of the male.     

Lignosulfonic acid blocks in vitro fertilization of macaque oocytes when sperm are treated either before or after capacitation

Lignin-derived macromolecules (LDMs) are biologically active compounds that affect a variety of cell-to-cell interactions including the inhibition of fertilization and embryo development in a number of nonmammalian species. The effect of ligno-sulfonic acid (LSA), a highly sulfonated LDM, on cynomolgus macaque sperm-oocyte interaction was evaluated with a zona pellucida binding assay and by in vitro fertilization (IVF). Sperm were treated with LSA (1.5 mg/mL) either before washing or after capacitation. Capacitation included centrifugation through 80% Percoll followed by 2 consecutive washes with medium, overnight incubation, and activation with dibutyryl cyclic adenosine monophosphate and caffeine. The zona binding assay was performed using immature oocytes that had adhered to the center of glass "binding chambers." The number of capacitated sperm that attached to the zona over a 3-minute period was recorded. Sperm attachment was significantly inhibited by LSA as compared to controls whether treatment occurred after capacitation (92.5%; P <.001) or before washing (82.5%; P <.001). When sperm were treated similarly with fucoidin, a sulfated polysaccharide known to inhibit sperm-oocyte interaction, sperm-zona binding was significantly inhibited by postcapacitation treatment but not by prewash treatment. Treatment of sperm with LSA consistently blocked fertilization over 4 IVF cycles both before washing and after capacitation. Fertilization rate for controls was 65% +/- 17%. No LSA-treated sperm were observed on the surface of lightly rinsed oocytes after 4 hours of coincubation. Localization of biotinylated LSA showed labeling over the entire sperm surface with the greatest intensity observed over the head and midpiece. LSA treatment had no effect on the percentage of motile sperm or quality of sperm motility. Due to the antifertility properties of this nontoxic molecule, LSA appears to have potential as a vaginal contraceptive.     

IVF-ET中未受精卵母细胞应用ICSI补救受精方法的结局评价

目的对体外受精-胚胎移植(IVF-ET)中未受精卵母细胞采用卵细胞胞浆内单精子注射(ICSI)作为补救受精方法后结局进行评价。方法回顾分析采用上述ICSI补救方法的11个周期病人,均以女方不孕因素为主进行IVF-ET治疗,在行常规受精18h后观察受精失败,即行ICSI再受精(Late ICSI),胚胎形成后行胚胎移植术,术后21d明确有无临床妊娠。结果11例病人共取出135只卵子,其中MII期107只,MI期23只,GV期5只,常规受精后只有2.80%受精(3/107)。随即对103只未受精卵行Late ICSI,术后受精率达到79.61%(82/103),卵裂率74.39(61/82),共移植25只胚胎,无一例临床妊娠。结论ICSI技术作为补救受精的方法能增加受精率,卵裂率,但不增加妊娠率;常规IVF-ET受精率低与男方精子活力低的关系有待进一步探讨。     

补救卵母细胞浆内单精子注射治疗的临床结局分析

体内精卵受精是一个复杂的过程,包括卵母细胞成熟、精子成熟、精子获能、精子透明带结合、配子融合、卵子激活和精子解聚等一系列过程,其中任何过程发生障碍都可能造成受精失败。在常规体外受精-胚胎移植（IVF—ET）周期中,常有一些周期存在受精完全失败的情况,具体原因在每一个受精失败周期中各不同。卵胞浆内单精子注射（ICSI）技术是直接将单一精子注射人卵细胞浆内,跨越了常规IVF时精卵结合等过程,