Difference in the seminal plasma protein expression in unexplained infertile men with successful and unsuccessful in vitro fertilisation outcome

Unexplained male infertility (UMI) is a condition in which routine semen analysis fails to detect subcellular sperm dysfunctions. In the present research, a comparative proteomics study of seminal plasma (SP) was conducted in men with unexplained infertility whose female partners had undergone in vitro fertilisation (IVF) treatment to find differences in the SP protein profile. Five UMI men with successful and eight with unsuccessful IVF outcome enrolled in this study. Two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) technique was used for protein separation. The differentially expressed proteins were identified using mass spectrometry. Results indicated that at least two different protein spots, including clusterin and epididymal secretory protein E1, were over‐expressed (1.5‐ and 2‐fold change, respectively, p < 0.05) while prostate‐specific antigen was downregulated (0.3‐fold change, p < 0.05) in the successful group as compared with the unsuccessful group. Considering the role of all three identified proteins in the sperm quality, the results of the present study introduced these proteins as new candidate biomarkers for success of IVF in UMI couples.     

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility – idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.     

Sperm chromatin stability and zinc binding properties in semen from men in barren unions

Sperm chromatin stability and zinc binding properties were studied in semen samples from 115 men living in barren unions. Of these men, 26% had a high proportion of swelling sperm, i.e. less than 80% sperm with stable chromatin after exposure to the detergent sodium dodecyl sulphate. From 2-67% of seminal zinc was bound to high molecular weight ligands of vesicular origin (HMW). This shows that, among infertile men, liquefied seminal plasma has huge variations in zinc chelating properties. The relationship between prostatic palpatory status, the proportion of abnormal sperm, the percentage zinc bound to HMW (HMW-Zn), the time between ejaculation and analysis and chromatin stability were studied. Samples with low chromatin stability were found more frequently in men with low HMW-Zn levels in semen. The proportion of stable sperm decreased in samples with prolonged exposure to seminal plasma. Neither the proportion of stable sperm heads nor the percentage zinc bound to HMW could be used to predict the future chances of the infertile men fathering children when studied 15-180 min after ejaculation. To differentiate between initial zinc-dependent stability and superstability developed in seminal plasma, other more sensitive methods must be developed.     

Effect of different culture techniques used to induce capacitation on the chromatin stability of human sperm

To validate an in-vitro bioassay for assessing chromatin stability of human sperm, 38 semen samples from infertile men were studied using sodium dodecyl sulphate, an anionic detergent which disorganizes only the cytoplasmic membrane. Assay sensitivity was 50 sperm, whilst the within- and between-assay variation, and the between-observer variation were found to be within the accepted range for this type of bioassay. The influence of different in-vitro treatments currently used in some clinical assisted fertilization programmes was evaluated: a destabilizing effect occurred in Grade I (stable) and Grade III (swollen) sperm. In the former, all treatments reduced stable sperm; in the latter, a significant (P less than 0.001) increase in swollen sperm was shown with two methods that used Ham's F-10 as culture medium. Different chromatin patterns found in the treated sperm suggest the possibility that the recovered samples could be modified compared to their status at the time of isolation.     

精子染色质扩散实验检测精索静脉曲张及不明原因不育患者精子DNA碎片

目的了解精索静脉曲张(VC)及不明原因不育患者精子DNA碎片的发生比例。方法改进的精子染色质扩散(SCD)实验分析精子DNA碎片。检测VC不育患者39例,不明原因不育患者57例。以生育健康成年男性32例为对照组。结果VC不育患者SCD小光晕和无光晕精子(精子DNA碎片)比值平均为(36.6±18.9)%,VC不育组明显高于对照组(12.1±5.2)%(P<0.001),而大光晕和中光晕精子比值VC不育组明显低于对照组(P<0.01);不明原因不育患者精子DNA碎片比值平均为(26.8±10.2)%,与对照组[(12.1±5.2)%]比较有显著性差异(P<0.001)。结论SCD实验表明,VC及不明原因不育患者精子DNA碎片比值增高。     

Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization (WHO) manual-based semen parameter's concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay (SCSA), a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection (ICSI).     

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。     

Sperm count and motility influence the results of human fertilization in vitro

In order to select sperm characteristics that can predict the outcome of in-vitro fertilization-embryo transfer (IVF-ET), semen samples delivered in conjunction with this treatment were studied carefully. We have analysed these data retrospectively in relation to the outcome of treatment. Ninety-one couples were treated for tubal infertility by IVF-ET. Fifteen women became pregnant. Sperm were isolated from semen using a swim-up technique and in most cases 40-80 x 10(3) (range 20-120 x 10(3)) motile sperm per ovum were used for insemination. The couples were divided into three groups: group A who achieved pregnancies, group B who achieved cleaved ova but no pregnancies, and group C who achieved no ova that were cleaved 48 h after oocyte recovery. Comparisons between these groups showed that some characteristics of the native semen samples and the swim-up preparations were significantly different: the sperm concentration (P = 0.001) and total sperm count (P = 0.003) in the native sample, the number of sperm recovered during 30 min of swim-up (P = 0.001), and the specific progressive motility of sperm in the swim-up preparation, both at the time of insemination and on each day, up to 5 days thereafter (P = 0.002-0.028). No pregnancy was achieved with a sperm concentration below 26 x 10(6) ml-1 in the native sample. Some of the sperm characteristics studied in this paper may be of value in the pretreatment evaluation for IVF treatment.     

Effect of high dose oral Kallikrein treatment in men with idiopathic subfertility: evaluation by means of in vitro penetration test of zona free hamster ova

High dose oral Kallikrein treatment (600 U Padutine per day) was given to 10 infertile men with poor semen quality. After 3 months of this treatment no pregnancies occurred, and no significant changes in routine semen characteristics were observed. Moreover, the capacity of the spermatozoa to migrate in vitro and to penetrate zona free hamster ova remained unchanged after Kallikrein ingestion. It is concluded that Kallikrein treatment was ineffective in these men.     

Intracytoplasmic sperm injection in cases with history of in vitro fertilization failure

Aim: To evaluate the effect of intracytoplasmic sperm injection (ICSI) in the management of cases with a history of conventional in vitro fertilization (IVF) failure. Methods: Two groups of patients, 19 with normal semen parameters and a history of IVF failure (metaphase Ⅱ oocytes: 0~30 %) and 28 with severe male factor infertility received ICSI technology during the same period. Ovarian stimulation was achieved by conventional procedure. Transvaginal ultrasound-guided oocyte collection was done 35~37 h after human chorionic gonadotrophin (hCG) injection. Only metaphase Ⅱ oocytes were selected for microinjection. Results: Fertilization was achieved with ICSI in all the patients. The fertilization rate (75.6 %±21.1 % vs. 73.9 %±19.2 %), cleavage rate (85.1 %±19.3 % vs. 82.7 %±22.1 %), clinical pregnancy rate per embryo transfer cycle (31.6 % vs. 28.6 %) and implantation rate per embryo (15.3 % vs. 14.4 %) did not differ significantly between the two groups. Conclusion: ICSI is a valuable method for     

原发性不育患者行体外受精-胚胎移植的病因及妊娠结局分析

目的对行体外受精助孕的原发性不育患者的病因进行分析,了解各病因所占的比例,并比较各组患者妊娠结局。方法对行体外受精的原发性不育患者按病因分为输卵管因素、男方因素、子宫内膜异位症(内异症)、排卵障碍和不明原因不育组,计算各组患者在总患者中的比例;比较各组卵泡刺激素(Gn)用量、获卵数、受精率、卵裂率、优质胚胎率、胚胎种植率、临床妊娠率及流产率。结果输卵管因素在原发性不育患者中比例最高(46.14%),其次是男方因素(30.48%);Gn用量男方因素组和排卵障碍组较少,与输卵管因素组比较有显著性差异(P<0.05);获卵数男方因素组最高,其次是排卵障碍组,与输卵管因素组比较无显著差异(P>0.05),内异症组获卵数最低,与输卵管因素组相比有显著性差异(P<0.05);排卵障碍组的受精率、卵裂率、优质胚胎率均最高,内异症组最低,但各组之间无显著性差异(P>0.05);胚胎种植率排卵障碍组最高(40.44%),输卵管因素组最低(18.44%);临床妊娠率内异症组和排卵障碍组分别为40.43%和36.67%,其次是男方因素组为34.50%,不明原因不育组为29.63%和输卵管因素组最低为26.86%;不明原因不育组的流产率最高为25%,内异症组和输卵管因素组各发生1例异位妊娠。结论输卵管因素在原发性不育患者中比例最高,其临床妊娠率低可能与内膜炎症和输卵管积水有关,子宫内膜异位症行预处理可获得较高的临床妊娠率。     

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

目的:使去透明带金黄地鼠的卵母细胞与携带乙肝病毒 DNA(HBV DNA)的人类精子体外受精,检测乙肝病毒基因(S 基因与 C 基因)在早期胚胎细胞中的复制与表达。方法:通过异种体外受精,由人类精子将乙肝病毒基因导入去透明带地鼠卵母细胞中;用聚合酶链反应(PCR)检测单细胞和2-细胞胚胎细胞的基因组中是否有 S 基因和前 C/C 基因;用逆转录聚合酶链反应(RT-PCR)研究上述基因是否在胚胎细胞中表达。以全长 HBV DNA 制备探针,与胚胎细胞制片进行荧光原位杂交(FISH),观察胚胎细胞基因组中是否有 HBV DNA整合。结果:PCR、RT-PCR 和 FISH 分析均在待测样本中获得阳性结果。结论:以精子为载体、携带到卵内的 HBV 基因能够在早期胚胎细胞中复制和表达,该结果为 HBV 有可能通过男性生殖细胞垂直传递给子代提供了直接证据。     

Evaluation of bovine cervical mucus penetration as a test of human spermatozoal function for an in vitro fertilization programme

Thirty-two couples participating in an in vitro fertilization (IVF) programme were evaluated as regards the prognostic value on fertilization of spermatozoal performance through flat capillary tubes filled with standardized midcycle bovine cervical mucus (Penetrak, Serono Diagnostics, Surrey, UK). A statistically significant correlation (P < 0.033) was observed between the distance travelled by the neat spermatozoa in the mucus and the % penetration of oocytes at IVF. There were also significant correlations between motility and progression (P < 0.004) and a borderline correlation between progression and the Penetrak results (P < 0.098). There was no significant difference between the Penetrak distances travelled between the 9 who conceived (33.4 mm) and the 23 who did not (29.9 mm). While the test does add to the knowledge of fertilization potential, the results extrapolated to a larger series would give false positive rates of 25% and false negative rates of 11%. The absence of a clear end point renders the Penetrak mucus penetration test insufficiently accurate to be used as a main measure of the male factor when advising for or against IVF therapy.     

Correlation analysis of the progesterone‐induced sperm acrosome reaction rate and the fertilisation rate in vitro

In this study, we aimed to investigate whether progesterone‐induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty‐six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone‐induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 ± 5.88% versus 9.66 ± 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = ?0.053, P > 0.01) or rate of high‐quality embryo development (r = ?0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = ?0.406, P > 0.01), fertilisation rate (r = ?0.148, P > 0.01) or progesterone‐induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone‐induced AR may not be used to indicate fertilisation rate.     

Comparison of sperm preparation methods: effect on chromatin and morphology recovery rates and their consequences on the clinical outcome after in vitro fertilization embryo transfer

The aim of this study was to evaluate the efficacy of swim-up, PureSperm gradient centrifugation and glass-wool filtration methods for semen preparation and to assess the possible enhancement of the quality of the subpopulation of spermatozoa in terms of sperm concentration, morphology and chromatin condensation. Moreover, to determine the effect of this semen processing technique on the clinical outcome after in vitro fertilization embryo transfer (IVF-ET). A total of 180 semen samples of patients' husbands who were undergoing IVF therapy were prepared by swim-up (G1, n = 60), PureSperm gradient centrifugation (G2, n=60) or glass-wool (G3, n=60) methods. Chromatin condensation was assessed by Chromomycin (CMA3), whereas sperm morphology was evaluated according to strict criteria. In all three semen processing methods, the percentage of chromatin condensed and morphologically normal spermatozoa was higher after semen processing in comparison with native semen samples. The proportion of normal chromatin condensed spermatozoa prepared in glass-wool filtration was significantly higher than that in swim-up (G.I, p=0.02) or PureSperm (G.II, p=0.001). In addition semen processing with PureSperm yields significantly a higher percentage of morphologically normal spermatozoa than swim-up (p < 0.001) or glass-wool method (p < 0.002). However, the fertilization, implantation and pregnancy rates, in turn were similar in all semen preparation methods. In conclusion, PureSperm gradient centrifugation yields a higher percentage of morphologically normal spermatozoa than shown in traditional swim-up or glass-wool filtration. However, the percentage of chromatin condensed spermatozoa was significantly higher after semen processing via glass-wool in comparison with the other two methods. Nevertheless, there were no significant difference in the fertilization, implantation and pregnancy rates of sperm prepared by means of swim-up, PureSperm or glass-wool filtration. Therefore, glass-wool filtration should be recommended as the first choice for semen preparation for Intracytoplasmic sperm injection (ICSI) technique as the natural selection is bypassed. Whereas, swim-up and PureSperm should be used for semen processing in IVF programme.     

The Importance of Sperm Motility for the Fertilization of Human Oocytes in vivo and in vitro

Motility behaviour of spermatozoa in 289 ejaculates was investigated by laser-Doppler-spectroscopy in the andrology-laboratory of the University Hospital for Gynecology and Obstetrics, Graz/Austria. Conventional spermiograms, performed on the same ejaculates, permitted comparisons between estimated motility and laser-measurements, comprising the following parameters: Total sperm motility, mean sperm velocity, percentages of progressive motile spermatozoa and mean progressive velocity. In order to elucidate the importance of sperm motility for the fertilization of oocytes in vivo and in vitro, ejaculates of six different groups of patients were evaluated. In vivo: 173 patients from the andrologic outpatient service (group A); 38 patients from the andrologic outpatient service, whose wives became pregnant (group B) and 20 donors with recently proved fertility (group C). In vitro: 8 patients, whose ejaculates failed to fertilize the oocytes (group D); 39 patients with successful fertilization (group E) and 12 patients with ongoing, clinical pregnancy of their wives after ivF (group F). In addition, motility behaviour was assessed in native ejaculates 24 hours after ejaculation, as well as changes in sperm motility after capacitation. Statistical evaluations of group comparisons revealed a good correlation between estimated results and laser measurements. Mean sperm velocity and progressive sperm motility are the most selective parameters within the group differences in oligozoospermic ejaculates. Besides motility, morphology is of importance in vivo as well as in vitro. Based on the results of measurements obtained in the different groups, the following guidelines for standard values in laser-Doppler-spectroscopy are proposed: Total sperm motility greater than 53%; mean sperm velocity greater than 42 microns/sec.; progressive sperm motility greater than 34% and mean progressive velocity greater than 68 micron/sec. If these values are not reached, medical treatment is recommended.     

Effect of freeze–thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men

Cryopreservation is known to impair sperm motility and decrease the fertilization rate by detrimental effects on acrosomal structure and acrosin activity. However, the consequences of cryopreservation on the integrity of the sperm nucleus, chromatin stability and centrosome are less clear. The present study was designed to determine the effect of the freeze-thawing procedure on chromatin condensation (aniline blue staining) and the morphology (strict criteria) and membrane integrity of human spermatozoa. The structural and functional characteristics of the sperm plasma membrane were measured by the eosin-test and hypo-osmotic swelling test which were done separately. Sperm cryopreservation was performed on semen samples from two groups of men classified as fertile (n = 20) and subfertile (n = 72), based on their reproductive history and semen analysis according to WHO guidelines. The mean percentage of condensed chromatin, morphologically normal spermatozoa and membrane integrity in all semen samples investigated (n = 92) decreased significantly (p = 0.0001) after freeze-thawing, in comparison to the value observed prior to freezing. By comparing the semen samples between fertile and subfertile patients, significantly (p = 0.0009) greater damage was demonstrated in the subfertile than in the fertile group. Furthermore, no significant difference was observed between the two groups with regard to the morphological alteration and structural as well as functional damage of the sperm membrane. In conclusion, the freeze-thawing procedure significantly affects chromatin structure and sperm morphology, especially in the head and the tail regions, and this may explain the lower fertilization rate and IVF/ICSI outcome when frozen-thawed spermatozoa are used. In addition, this study demonstrates that chromatin condensation is a sensitive parameter for the evaluation of cryodamage of semen samples from fertile and subfertile patients, though subfertile patients with very poor semen characteristics have yet to be studied. It is therefore recommended that chromatin condensation be used as an additional parameter for the assessment of sperm quality after freeze-thawing.     

66例男性不育患者采用自然周期IVF/M结合胞浆内单精子注射技术治疗的研究

目的探讨自然周期成熟卵母细胞结合未成熟卵母细胞体外成熟方法(natural cycle in vitro fertilization combined with in vitro maturation of immature oocytes,自然周期IVF/M)用于治疗男性因素引起不育的临床价值。方法66例适应证患者分成3组:少弱精子组、偶见活精子组和无精子症组[行经皮附睾精子抽吸术(PESA)、睾丸精子抽吸术(TESA)或睾丸切开取精术(TESE)],接受72个自然周期IVF/M,比较三组受精率和临床妊娠率。结果64例患者完成70个治疗周期。少弱精子组、偶见活精子组和无精子症组分别占完成周期数的54.3%、38.6%和7.1%,共获得卵母细胞812个,平均(11.3±5.2)个。卵母细胞成熟率为61.2%,正常受精率为82.2%,卵裂率为90.0%(377/419)。共26例患者获得临床妊娠,每取卵周期的妊娠率为36.4%(26/72),每移植周期的妊娠率为37.1%(26/70)。三组受精率分别为84.5%、78.9%和80.0%,三组间差异无统计学意义(P>0.05),临床妊娠率分别为39.5%,29.6%和60.0%,PESA/PESE和TESE组的临床妊娠率(60%)显著高于精液中偶见活精子组(29.6%,P<0.05)。结论自然周期IVF/ M技术对于治疗由于男性因素引起的不育如少弱精子症以及无精子症是一种简便有效的方法。     

首乌还精胶囊体外对人精子运动能力和受精能力的影响

目的 :研究首乌还精胶囊含药血清体外对人精子运动能力和受精能力的影响。　方法 :通过人精子与首乌还精胶囊含药血清共培养 ,观察首乌还精胶囊对人精子运动能力 (精子运动CASA分析 )、精子顶体反应和穿透去透明带仓鼠卵能力的影响。　结果 :含药血清加入共培养可显著提高人精子运动速度 [精子运动路径速度(VAP)、精子轨迹速度 (VCL)、精子前向运动速度 (VSL) ](P <0 .0 1)、精子头部侧置振幅 (ALH)和鞭毛摆动频率(BCF)及前向运动精子密度 (P <0 .0 5 ) ,精子顶体反应发生率 (P <0 .0 0 1) ,卵子受精率 (FR)及受精指数 (FI) (P <0 .0 1)。且含药血清刺激作用呈现出一定的量效关系。　结论 :首乌还精胶囊具有提高人精子运动能力和受精能力的作用。     

Acrosomal status of human spermatozoa after follicular fluid or calcium ionophore challenge in relation to semen parameters and fertilizing capacity in vitro

Summary.  The proportion of spermatozoa that undergo spontaneous acrosome reaction in vitro is relatively low. The proportion can be enhanced by incubation with either biological inducers such as follicular fluid or chemicals like calcium ionophore. It has been suggested that improper acro-somal reaction may be a cause of fertilization failure in vitro. The objectives of the present study were to assess the acrosomal status of human sperm following follicular fluid or calcium ionophore treatment and to analyse the relationship between spontaneous and induced acrosome reaction and fertilization rates in vitro by standard in vitro fertilization (IVF) technology. In all, 53 semen samples (22 normal and 31 subnormal) were studied. The effect of calcium ionophore A 23187 and follicular fluid was assessed using the fluorescence activated cell sorter. IVF results were evaluated in relation to the acrosome status of the sperm samples. Our results demonstrate that the effect of follicular fluid on the acrosomal status correlated positively with the effect obtained by the calcium ionophore (Pearson's correlation r = 0.45). A significantly higher percentage of maximal acrosome change ( P <0.02) was found in cases where fertilization occurred (19/27), than in sperm samples that did not achieve fertilization in vitro (8/27). The present finding that follicular fluid induced acrosome reaction can serve as a predictive tool which is as good as the ionophore treatment for assessing IVF outcome, supports the use of this method for clinical purposes.