Effect of progesterone on acrosome reaction, hypoosmotic swelling test, and DNA stability in human spermatozoa

The association between different sperm parameters, an in vitro effect of progesterone, has not been studied satisfactorily. In this article, the effect of progesterone on acrosome reaction (AR), plasma membrane integrity, and chromatin stability has been assessed in human spermatozoa with normal morphology and motility. Semen samples were obtained by masturbation from 25 patients. Two criteria of classification were utilized in this study: high motility group and normal morphology group incubated with progesterone. The effect of progesterone on AR, plasma membrane integrity, and chromatin stability in human spermatozoa with normal morphology and motility was realized. The results suggest that only the subpopulation of spermatozoa with normal morphology is able to undergo the progesterone-induced AR. It is possible that in the reproductive female tract it takes place a high selection of sperm with chromatin stability determined and optimal plasma membrane to undergo the AR prerequisite for the fecundation.     

人精子质膜孕激素受体研究

目的 :观察人精子表面孕激素受体 (PR)的定位及阳性表达率。　方法 :精子体外获能后 ,应用异硫氰酸荧光素标记的牛血清白蛋白 孕酮复合物 (P BSA FITC)染色 ,荧光显微镜观察及流式细胞术 (FCM )定量分析法 ,分别观察与孕酮 (P)结合精子形态及标记精子所占比例。　结果 :P BSA FITC染色精子的形态主要表现为 2种类型 :整个顶体区域或仅赤道区呈绿色荧光 ,顶体后区及尾部不着色。与P结合精子的百分率为 (30 2± 2 4 ) %。　结论 :人精子顶体表面有PR表达 ,且这种表达呈选择性     

Acrosome reaction in dog sperm is induced by a membrane-localized progesterone receptor.

The aim of this study was to investigate whether the dog sperm acrosome reaction can be induced by progesterone and whether the action of progesterone is mediated by binding of progesterone to a receptor on the sperm plasma. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Ten mM progesterone increased the acrosome reaction in viable spermatozoa over time from 3 +/- 1% at 0 hours to 69 +/- 8% at 6 hours (six dogs). In freshly ejaculated sperm from six dogs, P-BSA-FITC staining was observed in 13 +/- 1% of the viable, acrosome-intact cells, as characterized by bright fluorescence over the entire apical region. The proportion of P-BSA-FITC-stained, viable, acrosome-intact cells increased to 84 +/- 11% following 7 hours incubation in a low-calcium medium. In contrast, the majority (72 +/- 3%) of fresh epididymal sperm already demonstrated bright P-BSA-FITC staining. Apparently, epididymal spermatozoa already possess the progesterone receptor. The receptor is masked at ejaculation and subsequently gradually exposed.     

Selection of physiological spermatozoa during intracytoplasmic sperm injection

Sperm genomic integrity has a significant effect on intracytoplasmic sperm injection (ICSI) outcomes, especially post‐implantation. Spermatozoa selected based on motility and morphology do not guarantee the genomic integrity of spermatozoa. Nearly fifty percentage of spermatozoa in infertile men with normal morphology present different degrees of DNA fragmentation. However, capacitated or hyperactivated spermatozoa show lower degrees of DNA fragmentation. Therefore, selection of hyperactivated spermatozoa may improve ICSI outcome. Routinely, for ICSI, fast‐moving spermatozoa with A or B motility pattern are mainly selected for injection. The result of this study shows that in processed semen samples, hyperactivated spermatozoa are mainly observed in B motility pattern while, in viscous medium like polyvinylpyrrolidone (PVP), hyperactivated spermatozoa are mainly present in spermatozoa with C pattern of motility (nonprogressive). Therefore, we propose spermatozoa with C motility pattern which contains the main population of physiological or hyperactivated spermatozoa should be selected for ICSI.     

Effects of varicocelectomy on sperm DNA fragmentation, mitochondrial function, chromatin condensation, and apoptosis

The aim of this study was to evaluate conventional semen parameters (density, morphology, and progressive motility) and the flow-cytometric parameters of DNA fragmentation, mitochondrial membrane potential, phosphatidylserine externalization, and chromatin compactness in patients with varicocele before and after varicocelectomy. Thirty men (26.5 ± 3.2 years old, range 20-32 years) with oligoasthenoteratozoospermia and grade 3 left varicocele were selected (without other causes of male infertility). Each of them underwent sperm analysis and flow cytometric evaluation before and 4 months after subinguinal microsurgical varicocelectomy (SMV). After varicocelectomy, men had significantly higher sperm density, progressive motility, and normal forms compared with baseline. They also had a significantly lower percentage of spermatozoa with low mitochondrial membrane potential. After SMV, they showed a significantly lower percentage of spermatozoa with phosphatidylserine externalization, an early sign of apoptosis. Significantly decreased percentages of spermatozoa with abnormal chromatin compactness and spermatozoa with DNA fragmentation were found after SMV compared with baseline. Subinguinal microsurgical varicocelectomy improves sperm function in oligoasthenoteratozoospermia secondary to grade 3 left varicocele. Improvements are seen in conventional parameters and biofunctional parameters not routinely evaluated.     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.     

Sperm motility should be assessed in fresh sperm and after a sperm washing procedure.

A prospective study was carried out on semen samples from 118 consecutive unselected men attending our infertility clinic to determine whether sperm motility may be affected by seminal plasma. The incidence of asthenozoospermia as defined by fewer than 50% of spermatozoa with forward progressive motility in the untreated semen was 37.4%. This value was significantly reduced to 23% after washing and removing seminal plasma. Men with asthenozoospermia in untreated semen but normal in the washed sample had a percentage of normal sperm morphology and a percentage of swollen tails in the HOS test similar to those of controls, and higher than those of asthenozoospermics in both the untreated and washed sample. Sperm velocity was also significantly improved after the washing procedure. Spermatozoa selected by swim-down procedure and applied to a seminal plasma medium reduced sperm motility affecting negatively the HOS test. Sperm motility should be assessed after a sperm washing procedure, since seminal plasma contains constituents that decrease sperm motility without affecting membrane integrity.     

Evidence for association of sex hormone-binding globulin and androgen receptor genes with semen quality

The roles of androgen receptor AR(CAG)n gene polymorphisms and sex hormone-binding globulin SHBG(TAAAA)n gene polymorphisms on semen quality were studied. One hundred fourteen men were included in the study: 85 with normal sperm count and 29 oligospermic. The genotype analysis, on DNA extracted from spermatozoa, revealed five SHBG(TAAAA)n alleles with 6–10 repeats and 18 AR(CAG)n alleles with 12–32 repeats. The SHBG allelic distribution showed that in men with normal sperm count and motility, those with short SHBG alleles had higher sperm concentration than men with long SHBG alleles ( P  = 0.039). As concerns AR(CAG)n polymorphisms, men with short AR alleles had lower sperm motility compared to those with long AR alleles ( P  < 0.001) in both total study population and normal sperm count men. The synergistic effect analysis of the two polymorphisms revealed an association between sperm motility ( P  = 0.036), because of the effect of AR(CAG)n polymorphism on sperm motility. In conclusion, long AR alleles were found to be associated with higher sperm motility, while short SHBG alleles were associated with higher sperm concentration, supporting the significance of these genes in spermatogenesis and semen quality.     

Factors affecting fecundity among sperm donors: a multivariate analysis

The study was aimed at identifying the predictors of the male fertility potential among sperm donors. Fifty anonymous donors undergoing 683 intracervical insemination (ICI) cycles between January 2002 and December 2006 were retrospectively evaluated according to semen characteristics in terms of reproduction rate (RR). We used RR as a parameter to determine the fertility potential among sperm donors. The overall RR was 26.79%. There were no significant differences among low, mean and high RR groups with regard to most sperm routine parameters. However, the RR was notably higher in the sperm morphology of ≥18% than in the <18% group (26.2% versus 19.4% respectively; P < 0.01). Both post-thaw total motility and progressive motility were proportional to RR (P < 0.01). Differences in RR were seen when the percentage of propidium iodide-negative spermatozoa was ≥45% (26.2% versus 16.4% respectively; P < 0.01) and DNA fragmentation index (DFI) was <8% (37.5% versus 17.9% respectively; P < 0.01) in post-thaw samples. Using stepwise linear regression analysis, the percentage of normal morphology, post-thaw progressive motility, PI-negative spermatozoa, DFI had the maximum power to predict the donor fecundity in ICIs. Conclusion: Both the integrity of plasma membrane and DNA in spermatozoa are crucial factors affecting the fecundity of sperm donors. Therefore, the addition of some of these new tests to routine semen analysis could significantly improve the recruitment of sperm donors and the clinical pregnancy rate of anonymous donors.     

Relationship between semen characteristics, a-glucosidase and the capacity of spermatozoa to bind to the human zona pellucida

Unfertilized oocytes from an in-vitro fertilization programme were stored in different saline solutions and then utilized in a zona binding assay (ZBA). The four saline solutions tested were identical with regard to the capacity of the zona to bind sperniatozoa provided by healthy donors. Spermatozoa from 150 infertile patients were tested in the ZBA. The number of spermatozoa bound to the zona correlated positively with sperm concentration, the percentage sperm motility and the percentage of morphologically normal spermatozoa. The population was then divided into two groups according to the level of α-glucosidase activity, an epididymal marker. The average number of spermatozoa bound to the zona was diminished in the group with low α-glucosidase activity, even when considering strictly equivalent classes of sperm concentration, motility and morphology, respectively.     

Morphology of spermatozoa in infertile men with and without varicocele

This study was carried out to evaluate the morphology of spermatozoa in infertile men with and without varicocele. A series of 285 ejaculates were fully evaluated for seminal volume, sperm count, motility, and morphology, and classified into fertile (165 subjects), infertile without varicocele (93 subjects) and infertile with varicocele (27 subjects). Sperm morphology was classified by multiple entry criteria and recorded as normal, abnormal with head, midpiece, or tail single anomaly or abnormal with simultaneous multiple abnormalities. Semen volume was almost identical in the three groups. Among the infertile men, sperm count was lower in those having a varicocele, but conversely those with varicocele had a higher percentage of motile spermatozoa, higher percentage of spermatozoa with forward movement and higher sperm velocity. There were higher proportions of spermatozoa with abnormal morphology, total number of anomalies, and multiple anomalies in infertile men, both with and without varicocele, than in fertile men. The percentage of abnormal spermatozoa was higher in infertile men with varicocele than in those without varicocele. The pattern of sperm morphology differed between the infertile and the fertile group, and with the presence or absence of varicocele. In the presence of varicocele, only the incidence of elongated (tapered) forms was significantly increased.     

镉离子对公牛精子功能损伤的机制

目的:重金属比如镉(Cd)作为工业污染物广泛分布在环境中,研究人员已经鉴定了这些污染物能影响男性生殖系统。本文研究的目的是测试Cd在10~1000μmol/L的浓度范围在体外对荷斯坦(Holstein)公牛精子膜和DNA完整性、活动率和精子顶体胞吐能力的影响。方法:用PBS处理公牛精液样本后进行精液分析。脂质过氧化试验评估精子膜完整性。明胶消化试验测定公牛精子顶体胞吐作用的能力。单细胞凝胶电泳(SCGE)检测单个细胞内DNA断裂和不耐碱的破坏。结果:脂质过氧化(LPO)显著增加,表明了Cd对精子膜完整性的破坏作用。这种影响在Cd浓度为1000μmol/L时特别明显。LPO与活动精子百分率之间呈负相关(r=-0.94,P<0.001)。明胶消化试验表明Cd引起公牛精子顶体胞吐作用的百分率下降。发现在LPO率和消化环百分率之间呈负相关(r=-0.97,P<0.001)。彗星试验获得的数据表明Cd能诱导精子核中的DNA断裂。接近93%的DNA损伤为双股断裂。LPO氧化率与DNA断裂百分率之间的相关性为0.95(P<0.001)。结论:总体上,Cd诱导公牛精子膜损伤、活动率降低、DNA断裂、以及顶体反应率降低而导致精子功能损伤。进入雄性性腺和精浆的Cd可能对动物精子产生了破坏作用。     

Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle

In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.     

The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas

This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (±SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation.     

Recovery and cryopreservation of Spanish ibex epididymal spermatozoa

A Tris-citric acid-glucose (TCG) diluent containing low concentrations (6%) of egg yolk, and a TCG extender containing lactose (without egg yolk), were compared for use in the cryopreservation of Spanish ibex (Capra pyrenaica) epididymal spermatozoa. To optimize the collection of epididymal spermatozoa, two spermatozoa recovery methods were tested: i) by using small cuts in the cauda epididymides and ii) by the application of air pressure (from a syringe) inside the vas deferens. The percentage of viable spermatozoa recovered was lower (P < 0.05) with the air pressure method. No significant differences were seen in the efficacy of the two diluents as determined by percentage viability of thawed sperm, membrane integrity (as determined by the hypo-osmotic swelling test), or acrosome integrity. The use of the TCG-lactose medium strongly reduced sperm motility (P < 0.001). The sperm samples that had been diluted with TCG-6% egg yolk extender showed a greater incidence (P < 0.05) of morphological abnormalities. TCG-lactose alone, does not well preserve motility when cryopreserving Spanish ibex epididymal spermatozoa.     

Computer-assisted assessment of sperm morphology: comparison with conventional techniques

The aim of the present study was to compare conventional and computer-assisted morphology assessment of spermatozoa. Sixty-two semen samples from patients undergoing in vitro fertilization (IVF) and 40 samples from patients undergoing an intracytoplasmic sperm injection (ICSI) were studied using both techniques. The percentage of normal spermatozoa found was closely correlated between the techniques (r=0.788, p < 0.0001). The intra-operator variation was low for both techniques but the inter-operator variation was much higher with the conventional than with the computer-assisted method (coefficient of variation = 0.43 vs. 0.08, respectively, for conventional and computer-assisted assessments). The percentage of spermatozoa with normal morphology, as well as sperm motility, was significantly enhanced after PureSperm preparation, whatever the method used for assessment. In the IVF study, fertilization rate was poorly correlated with sperm morphology using both methods. However, combined with motility, morphology assessed with the computer allowed discrimination of two groups of patients with significantly different fertilization rates (30.5 +/- 5.4% vs. 63.1 +/- 5.4%, p < 0.0001). In contrast, the fertilization rate in ICSI was influenced neither by sperm morphology nor by motility. In conclusion, computer-assisted assessment of sperm morphology has a slightly better predictive value for ART than conventional assessment, but above all is much more reproducible, allowing standardization.     

Decline of semen quality among 10 932 males consulting for couple infertility over a 20-year period in Marseille,France

Semen from 10 932 male partners of infertile couples was analysed and sperm parameter trends were evaluated at the Reproduction Biology Laboratory of the University Hospital of Marseille (France) between 1988 and 2007. After 3–6 days of abstinence, semen samples were collected. Measurements of seminal fluid volume, pH, sperm concentration, total sperm count, motility and detailed morphology of spermatozoa were performed. Sperm parameters were analysed on the entire population and in men with normal total numeration (≥40 million per ejaculate). The whole population demonstrated declining trends in sperm concentration (1.5% per year), total sperm count (1.6% per year), total motility (0.4% per year), rapid motility (5.5% per year) and normal morphology (2.2% per year). In the group of selected samples with total normal sperm count, the same trends of sperm quality deterioration with time were observed. Our results clearly indicate that the quality of semen decreased in this population over the study period.     

Seasonal variation and age-related changes in human semen parameters

Although semen quality has been discussed extensively with regard to age and season in the andrology literature, the results vary and firm conclusions are still outstanding. To investigate seasonal and age-related variations in human semen parameters, we analyzed data that were collected from an andrology clinic population. We performed a retrospective review of 551 semen analysis records collected from 1989 to 2000 from the Vincent Memorial Andrology Laboratory at Massachusetts General Hospital. Semen volume, sperm concentration, total sperm count, motility, total motile sperm, and morphology significantly decreased as age increased. In addition, as age increased, the percentage of sperm with tail defects increased. Sperm concentration was significantly higher in winter (mean 157.9 million/mL) than in fall (mean 119.1 million/mL) (P <.05). The mean percentage of sperm with normal morphology was significantly higher in winter (9.2%) than in summer and spring (7.0% and 7.5%, respectively; P <.05). The mean percentage of sperm with head defects was significantly higher in fall and summer (74.0% and 72.3%, respectively) than in winter (68.6%; P <.05). Seasonal variations were found in sperm concentration and morphology, with higher sperm concentrations in winter than in fall, and a greater percentage of sperm with normal morphology in winter than in spring and summer. Sperm concentration was lowest in the fall, whereas the percentage of sperm with normal morphology was lowest in summer. Semen volume, sperm concentration, total sperm count, motility, total motile sperm, and morphology decreased as age increased.     

Comparison and evaluation of capacitation and acrosomal reaction in freeze‐thawed human ejaculated spermatozoa treated with L‐carnitine and pentoxifylline

Cryopreservation is used to preserve the spermatozoa; however, it leads to a reduction in sperm quality. L‐carnitine (LC) influences sperm motility and preserves the sperm membrane and DNA integrity. The objectives of this study were to evaluate the protective effects of LC on the membrane integrity of normal human spermatozoa and compare it with pentoxifylline (PT) during cryopreservation. Thirty normal semen samples, prepared by swim‐up procedure, were divided into three aliquots: a control without any treatment and two experimental aliquots that were incubated in PT or LC for 30 min. All aliquots were cryopreserved and thawed after 48 hr. To evaluate the percentages of intact, acrosomal‐reacted and capacitated spermatozoa, lectin histochemistry and flow cytometry were performed by wheat germ agglutinin, peanut agglutinin and Con A. Statistical analyses were performed using ANOVA. LC supplementation elevated the percentage of noncapacitated spermatozoa compared with control and PT‐treated samples and the percentages of acrosomal intact spermatozoa compared with PT‐treated samples. PT pre‐treatment improved the motility but not membrane integrity. LC supplementation reduced the percentages of acrosomal‐reacted spermatozoa compared with the control and PT‐treated samples. Although LC did not improve motility, it protected the plasma membrane and acrosomal integrity. Therefore, LC may be the superior choice compared to PT for maintaining the sperm integrity.