Evaluation and assessment of semen for IVF/ICSI

Evaluation and assessment of semen is very important for both diagnosis of male infertility and selection of patients for treatment with IVF or ICSI. In standard IVF, sperm function is essential for normal fertilization: sperm must be able to bind to zona pellucida (ZP), undergo the acrosome reaction and penetrate the ZP and fuse with the oolemma before fertilization takes place. In contrast, most sperm functions are not required for fertilization in ICSI since sperm bypass the ZP and oolemma by injection of a single sperm directly into cytoplasm of oocyte. Therefore, the clinical decision on treatment of patients with either IVF or ICSI is mostly dependent on results of sperm tests. However, conventional semen analyses do not provide accurate information about sperm fertilizing ability since many patients with subtle sperm defects can not be detected. More advanced sperm function tests are required to detect sperm defects that may lead to failure of fertilization in standard IVF. In the last 15 years w     

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。     

Body mass index is not associated with sperm-zona pellucida binding ability in subfertile males

Lifestyle factors, such as weight and nutritional status may affect male fertility, including sperm fertilization ability. The objective of this retrospective study was to evaluate the association between body mass index （BMI） and sperm-zona pellucida binding ability assessed according to the zona binding （ZB） test, which has been described to be a relevant diagnostic tool for the prediction of in vitro fertilization （IVF） ability. Three hundred and six male patients from couples diagnosed with primary idiopathic or mild male factor infertility were included. Correlations between BMI and semen parameters according to ZB test indices were assessed, together with frequencies of positive and negative tests across the BMI categories. In this selected population, BMI was not related to conventional semen parameters or sperm quality assessed according to the ability of spermatozoa to bind to the zona pellucida. The previously described poor outcomes of IVF procedures in cases of male obesity could be due to other sperm defects, such as alterations of sperm capacitation or acrosome reaction. The link between male BMI and biological outcomes during IVF procedures, such as fertilization rates, should be further evaluated.     

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。     

New aspects of sperm-zona pellucida binding

Summary.  Sperm-zona pellucida binding tests have become a widely used diagnostic application for clinicians to obtain guidance in so far as the therapeutic approach of the subfertile couple is concerned. Expanding the oocyte sources is imperative to ensure the consistent use of spermzona binding assays. Sources include oocytes derived from post-mortem ovaries, inseminated non-fertilized IVF oocytes and recycled hemizonae. Identification of specific gamete dysfunction is one of the most formidable tasks and fertilization disorders due to defective sperm-zona pellucida interaction are relatively common in the clinical practice, thereby emphasizing the importance of sperm-zona binding tests as diagnostic/predictive tests. Independent publications from Norfolk (USA), Melbourne (Australia), Tygerberg (South Africa) and Israel of highly comparable results confirm that sperm-zona binding tests are good predictors of fertilization. Studies using solubilized human pellucida recently evaluated the influence of solubilized human pellucida on spermatozoa during the capacitational process and subsequent sperm-zona binding. Involvement of G protein and carbohydrate moieties in sperm-zona pellucida binding emphasized the biological and biochemical properties of lectin and have afforded much weight to their employment as membrane probes to evaluate cell surface components. Attention has been focused on the alterations of sperm surface receptors (oligosaccharides) during the differential pathway, epididymal transit and capacitation.     

Differential effects of polysulphates between mouse and hamster during in vitro fertilization

The interaction between zona pellucida polysulphates and sperm receptors appears to be a widespread mechanism used by mammals during gamete interaction. In this work, the effect of heparin on binding, penetration and fertilization of mouse and hamster oocytes was assessed. We found that heparin inhibited oocyte penetration and fertilization in both species. Heparin as well as fucoidan (a fucose-sulphate polymer) inhibited the proteolytic activity of acrosomal enzymes in both species. Our results suggest that zona pellucida penetration in both species may be modulated by polysulphates acting on either the proteolytic rate of degradation of the zona and/or its interaction with acrosome-reacted sperm (secondary binding).     

Correlation between sperm penetration into the human zona pellucida and in vitro fertilization rates

Summary.  Sperm penetration into the zona pellucida of unfertilized oocytes, and its correlation with in vitro fertilization rates of the sibling oocytes, were assessed. This was performed in order to evaluate the prediction rate of the sperm penetration test into the zona pellucida. Unfertilized oocytes ( n =1872) from 371 cycles were pipetted through a microcapillary, and the remaining sperm cells penetrating the zona pellucida were counted. The mean (±SD) number of spermatozoa that penetrated the zona pellucida of unfertilized oocytes was 12.9±16.37. A significant correlation was found between the fertilization rate and the mean number of spermatozoa that penetrated into the zona pellucida of the unfertilized sibling oocytes (r = 0.48; P < 0.001), or the percent of unpenetrated zonae pellucidae in a cohort (r= —0.43; P < 0.001). However, a distinct variation in the number of spermatozoa that penetrated into the zona pellucida was detected. A step-wise regression analysis proved the number of spermatozoa penetrating the zona pellucida to be more predictive for fertilization rates than the variable of percent of unpenetrated zonae pellucidae. The results imply that although there is interdependence between penetration into the zona pellucida and fertilization rate, the predictive value of sperm penetration test for prognosis and future management after the first in vitro fertilization attempt, is limited.     

Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility

Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.     

Electron microscopic evidence on the acrosomal status of bound sperm and their penetration into human hemizonae pellucida after storage in a buffered salt solution.

The hemizona assay (HZA) was developed to evaluate sperm binding potential using microbisected human zona pellucida. In this study, eight human oocytes stored in a buffered salt solution for 60 days were bisected into two identical hemispheres (hemizonae) and coincubated with the spermatozoa from a fertile man. All evaluated spermatozoa were tightly bound to the outer surface or had begun penetration into the zona pellucida. The hemizonae with bound spermatozoa were prepared and fixed for transmission electron microscopy (TEM) using standard techniques. Among the 108 sperm bound to the zone we were able to evaluate 25 by TEM. Twenty (80%) of the zona bound spermatozoa were partially or completely acrosome reacted, while six (20%) of the zona bound sperm had intact acrosomes. Acrosome intact, partially acrosome reacted and completely reacted spermatozoa were observed within the zona. Penetration pathways or tunnels were seen within the zona matrix. The results illustrate, that typically spermatozoa tightly bound the human zona pellucida show induction of the acrosome reaction. Importantly, following storage of human eggs in salt solution (buffered to 7.4), the zona pellucida retain their biological and functional characteristics for at least 90 days.     

Sperm penetration through the human zona pellucida as a predictor of in vitro fertilization

The aim of this study was to determine the predictive value of sperm penetration into the perivitelline space of human cadaveric oocytes on in vitro fertilization outcome. Forty-two patients with tubal infertility undergoing ovarian stimulation with gonadotropin for in vitro fertilization and embryo transfer participated in the study. The number of spermatozoa bound to the human zona pellucida, the percentage of cadaveric oocytes with one or more spermatozoa in the perivitelline space, and the in vitro fertilization outcome were evaluated. Spermatozoa from 37 of 42 patients were able to penetrate the perivitelline space of cadaveric oocytes as well as to fertilize human oocytes in vitro. In three individuals, no penetration of the perivitelline space of cadaveric oocytes was observed and no in vitro fertilization was detected. Only two patients were able to fertilize the couple's oocytes without penetration of the cadaveric oocytes. Based on these results the specificity and the sensitivity of the assay to predict in vitro fertilization was 100% and 94.1%, respectively. Accordingly, these results suggest that sperm-zona penetration is a useful bioassay to predict male fertility potential in IVF outcome.     

In vitro tests for essential sperm functions using the phyto-oestrogen genistein as a test substance

Sperm motility, binding of spermatozoa to the zona pellucida and induction of the acrosome reaction are prerequisites for successful oocyte fertilization. Examination of the physiological and nonphysiological effects of particular compounds on sperm functions requires high-quality in vitro test systems. In this short methodological overview, a reliable combined in vitro test system with bovine gametes is described. The purpose of the study was to evaluate whether aliquots of pooled post-thaw spermatozoa are suitable for examination of environmental substances that affect essential sperm functions. The combined test system includes a number of known methods for the assessment of sperm vitality and motion parameters, acrosomal status, inducibility of acrosome reaction and sperm zona pellucida binding. First observations indicate that genistein inhibits the induction of acrosomal exocytosis and binding of spermatozoa to the zona pellucida. Motility parameters and the viability of bovine spermatozoa were not affected by this substance. It is concluded that genistein, a phyto-oestrogen which is abundant in several plants, can be used as a test substance for the evaluation of effects upon essential bovine sperm functions in vitro.     

体外受精中的受精失败

受精是雌雄配子结合形成合子的过程,它标志着一个新生命的开始,是自然界最神秘和复杂的事件之一.受精失败（Total fertilization failures, TFF）特指在体外受精（in vitro fertilization, IVF）培养过程的一定时段内[包括常规受精和卵胞浆内单精子注射（ICSI）],精卵没能结合为合子,形态学上没有看到精原核和卵原核形成的状态.     

In vitro fertilization failure: identification of gamete defects by investigation of sperm-zona pellucida binding capacity of unfertilized oocytes

Summary. The objective of the study was to determine whether fertilization failure was due to spermatozoal or oocyte factors. Twenty-five unfertilized oocytes from 12 IVF/GIFT couples showing total or partial fertilization failure were evaluated for sperm zona binding potential under hemizona assay (HZA) conditions. Hemizonae were separately incubated with a sperm sample from the husband and that of a fertile control. Tight sperm binding to hemizonae was assessed. First, among the 12 patients, results showed a possible zona defect thought to be the cause of fertilization failure in five cases. Second, in two cases, fertilization failure was possibly caused by poor sperm binding potential of spermatozoa. Third, in two cases, fertilization failure was possibly caused by an oocyte defect, and fourth, three cases showed a mixture of possible causes. The results stress the need to develop a sequential analytic programme for those couples with repeated total or partial fertilization failure.     

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。     

The zona pellucida 'receptors' ZP1, ZP2 and ZP3.

The male component that is necessary for successful reproduction depends on a large variety of biological processes working in concert. The sperm-egg interaction occurs through complementary molecules and is an obligatory process for successful fertilization. However, this complex phenomenon and its molecular mechanisms remain to be fully understood. The oocyte is protected by the zona pellucida, a network of various proteins which encloses the oocyte. Depending on the species, the zona pellucida consists of different glycoproteins that are proposed to function as 'receptors' for spermatozoa. In the mouse, ZP1 is the homodimeric filament crosslinker, held together by intermolecular disulphides. ZP2 is the 'secondary receptor', which is cleaved by egg proteases after egg activation. The mouse ZP3 protein appears to be the 'primary receptor', which is responsible for species-specific binding of spermatozoa to the oocyte and the induction of the acrosome reaction. To localize zona pellucida protein and to evaluate the function of ZP2 and ZP3, polyclonal antisera were raised against synthetic ZP2 or ZP3 peptides which are specific for human or for mouse zona pellucida proteins. It could be demonstrated that anti-synthetic peptide antisera detected their respective zona pellucida proteins in immunoblots, ovary sections and native hemizonae pellucidae. Functional assays with anti-ZP3 synthetic peptide antibodies revealed that the antisera did not inhibit sperm-zona pellucida binding, whereas one of the antisera against synthetic ZP2 peptides significantly inhibited binding of spermatozoa to the zona pellucida.     

Sperm-zona pellucida interaction in cynomolgus and rhesus macaques.

These experiments were carried out to establish and validate an in vitro system for studying macaque sperm-zona pellucida interaction. Sperm of rhesus and cynomolgus macaques were capacitated in vitro and incubated with cryopreserved zonae pellucidae. Homologous gamete incubations were tested, as well as cross-species combinations. Approximately 25% of macaque sperm bound to the zonae acrosome reacted within 1 minute of gamete coincubation, although the percentage of acrosome reactions in the sperm suspension was less than 1%. There was a small but consistent increase in the percent of acrosome reactions of zona sperm after an additional hour of incubation in sperm-free media. Similar results were obtained in the cross-species experiments, suggesting that zonae from the two macaque species can be used interchangeably in sperm-zona binding assays. Differences in the physiologic characteristics of the sperm of the macaque species were demonstrated. Cynomolgus sperm required activation with caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) in order to bind to the zonae. Rhesus sperm were able to bind to the zonae and acrosome react in the absence of activators, although both sperm binding and percentage of acrosome reactions increased with the addition of activators. Large numbers of sperm from both macaque species bound to the zonae of hamster oocytes after treatment with activators, but the bound sperm did not acrosome react. These experiments demonstrate the importance of evaluating the acrosomal status of sperm when sperm-zona binding assays are performed with macaque gametes.     

精子胞膜脂质及其在受精中的作用

通过受精来完成精子与卵子融合并产生二倍体合子(受精卵)是有性生殖的标志,在这一过程中精子胞膜脂质发挥了重要作用。精子在成熟过程中,由于细胞器的丢失和DNA转录的终止,精子最终停止合成膜脂质、蛋白质,并且囊泡转运也终止。然而精子胞膜脂质成分却仍发生着复杂变化,并籍此在精子获能、与卵子透明带结合、顶体反应以及精子与卵子膜融合等过程中均起着重要作用。本文就精子膜脂质的组成、结构特点、过氧化作用、代谢及其在受精中的作用进行综述。     

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.     

Clinical importance of a micro-assay for the evaluation of sperm acrosome reaction using homologous zona pellucida

This study aimed to develop an acrosome reaction assay using microvolumes of solubilized human zonae pellucidae among 35 couples attending an in vitro fertilization programme. The sperm morphology of the men was classified as g-pattern (5-14% normal forms) and/or normal pattern (> 14% normal forms). All the couples had a history of repeated poor or failed in vitro fertilization rates from previous attempts. A zona-induced acrosome reaction test was performed using homologous 0.25 zona pellucida microl-1 incubated with spermatozoa to induce the acrosome reaction. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between zona-induced and spontaneous acrosome reaction spermatozoa. The results indicated that microvolumes of solubilized human zona pellucida could successfully be used to determine the acrosome reaction status of spermatozoa. The results were compared with in vitro fertilization rates of metaphase II oocytes, and analysed with the receiver operating characteristics curve. Receiver operating characteristics analyses divided the patients into two groups: i.e. zona-induced acrosome reaction < 15% and > 15%. The sensitivity and specificity for zona-induced acrosome reaction results versus fertilization were 93% and 100%, respectively. The correlation coefficient between zona-induced acrosome reaction and in vitro fertilization was r = 0.94 (P < 0.0001). Zona-induced acrosome reaction data can be used as an indicator for fertilization failure, thus helping clinicians to refine the therapeutic approach for infertile couples prior to the onset of the treatment.     

Inhibition of sperm-zona pellucida binding by a 55 kDa pig sperm protein in vitro

This study was conducted to evaluate the role of a 55 kDa pig sperm protein on the oocytesperm binding process, and its location in situ. For this purpose, in vitro matured oocytes were incubated with isolated and purified protein, and incubated with capacitated spermatozoa. In addition, capacitated sperm were incubated with anti-55 kDa antiserum and later with mature oocytes. Immunolocalization assays were performed using non-capacitated, capacitated and acrosome reacted sperm, which were incubated independently with anti-55 kDa protein antibodies and analyzed under fluorescence light microscopy. The 55 kDa protein concentrations correlated negatively with the amounts of sperm bound to the zona pellucida (ZP); the presence of the anti-55 kDa protein totally inhibited this binding. The immunolocalization assays revealed that fluorescence was located preferentially at the apical edge of the head in capacitated sperm, but not in acrosome reacted sperm. It would appear that the 55 kDa protein binds specifically to the oocyte ZP, and that it may be responsible for primary gamete binding during fertilization.