Comparison of nitric oxide production motion characteristics of sperm after cryopreserved in three different preparations

Three ml of semen collected from 20 volunteers were divided into three aliquots: a) semen cryopreserved for 48 hr then swim-up method; b) using swim-up method then cryopreserving for 48 h; and c) using 45%-90% two layers PureSperm to prepare sperm then cryopreserved for 48 h. Motility characteristics were evaluated before cryopreservation and after thawing. NO was measured using the chemiluminescence method after centrifugation. The recovery rate and motion parameters were the highest in the swim-up group. NO produced in the swim-up group was significantly lower than the PureSperm group, and the PureSperm group was lower than the semen treated with cryoprotectant group. Best semen quality was achieved due to the decreased NO production.     

Computer Assisted Measurement in Normal and Pathological Human Semen, Fresh and Post Swim-up Technique/Computergestützte Messung im normalen und pathologischen Sperma des Menschen, Frisch- und post-swim-up-Technik

A total of 429 semen samples were studied. Two hundred and twenty-nine samples with normal characteristics were processed following a basic procedure; the remaining 200 were normal and pathological samples that were analyzed pre and post swim-up. Semen specimens were allowed to liquefy for 30 minutes and sperm count, motility, velocity and linearity were determined using the Cellsoft Automatic Semen Analyzer. In normal patients, a significant increase of motility, velocity and linearity (p less than 0.001) post swim-up, was observed. Sperm recovery in this group was 13.9 +/- 1% of the whole motile sperm population. In the polyzoospermic group, recovery of motile spermatozoa post swim-up was significantly decreased as compared with the normal group (p less than 0.001). In the asthenozoospermic group (plus either hypospermia or hyperspermia, or with more than 30% of motile spermatozoa or less than 30% of motility), no variations of velocity or linearity as compared to the normal group were observed. In all the pathological groups studied, a significant increase in velocity and linearity (p less than 0.001) post swim-up, was observed.     

Comparison of centrifugation- and noncentrifugation-based techniques for recovery of motile human sperm in assisted reproduction

To compare standard density gradient centrifugation sperm preparation with a novel non-centrifugation-based dual-chamber capillary dish in efficiency for motile human sperm separation, approximately 3 mL fresh ejaculate specimens was obtained from 21 men (median age = 32 years. range 26-42 years) undergoing infertility evaluation. For each specimen, half of the sample was processed with a standard 45%/90% density gradient preparation (PureSperm. Nidacon International, Gothenburg, Sweden) followed by semen analysis. The other half was incubated in the Zech glass capillary dish (Astromedtec, Salzburg, Austria) consisting of 2 concentric wells overlaid by a U-ring and coverglass. After approximately 3 h, a 1-mL sample was taken from the central chamber and analyzed. Percentage motile sperm recovery, absolute (motile) cell number, and path velocities were compared for spermatozoa obtained from both methods. Both techniques reduced overall sperm concentration while enriching specimens with more motile spermatozoa. A trend towards higher % recovery of motile spermatozoa (p = .264) was observed with the Zech device, but at a cost of fewer absolute numbers of higher velocity cells (p = .004). The Zech device, therefore, localized a very small population of motile sperm without exposure to centrifugation stress, which has been considered potentially harmful to spermatozoa. This technique could theoretically improve efficiency by reducing time required to identify motile cells in in vitro fertilization where intracytoplasmic sperm injection is planned. However, refinements in incubation interval and suspension volumes are needed before this technique can be considered comparable to the density gradient method in recovering sperm for use in intrauterine insemination.     

Efficacy of the density gradient centrifugation method in eliminating sperm with aneuploidy

The aim of this study was to evaluate the efficacy of the PureSperm density gradient centrifugation on the selecting sperm with less chromosomal aneuploidy. Semen samples were obtained from 30 infertile men with teratozoospermia and 15 fertile men with normal semen parameters. The frequencies of numerical chromosomes aberrations were simultaneously identified in neat semen and in the different fractions of the density gradient centrifugation from the same samples. Using a triple colour FISH, we show that patients with severe teratozoospermia have a significantly increased frequency of chromosomal abnormalities in their neat semen compared with normozoospermic men (P < 0.001). The mean sperm motility and sperm morphology were improved significantly after semen processing with three layers PureSperm gradient compared with whole semen (P < 0.001). In addition, aneuploidy frequencies were lower in specimens enriched by the gradient centrifugation compared with unprocessed semen. Significant differences were observed in the disomy rates for the autosome and for either sex chromosome between the neat semen and the different PureSperm fractions (P < 0.001). In conclusion, our study shows that semen processing by density gradient centrifugation is very efficient in reducing sperm with aneuploidy and diploidy.     

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.     

Influence of sperm processing on the fertilizing capacity and recovery of motile sperm from thawed human semen

Artificial insemination programs now rely almost exclusively on frozen semen preparations as their source of sperm. Unfortunately, several reports indicate that conception rates using frozen-thawed semen are inferior to freshly ejaculated specimens. The present study was designed to investigate the fertilizing capacity of sperm and the recovery of motile sperm from thawed semen following different sperm processing methods. Washed resuspended pellets contained sperm with at least the same fertilizing potential as sperm from swim-up techniques. However, the recovery of motile sperm from the pellets was more than 3 times greater than from the swim-up techniques. Percentage motility, progressive velocity, and amplitude of lateral head displacement were generally higher in the sperm from swim-up techniques than in the sperm from the pellets, despite equivalent fertilizing potentials. It can be argued from these results that washed resuspended sperm preparations provide a significantly greater number of motile sperm without a loss in fertility when compared to sperm from swim-up techniques. Accordingly, this study raises questions about the use of the sperm swim-up as a procedure for processing thawed semen for use in intrauterine insemination in which maximal numbers of motile sperm are required. It also demonstrates the need to identify new methods for processing frozen-thawed specimens for assisted reproductive procedures.     

两种精子处理方法的卵细胞胞质内单精子显微注射结局的比较

目的:评价密度梯度离心和改良上游法两种处理活动精子的分离方法在卵细胞胞质内单精子显微注射(ICSI)中的效果,从而指导临床应用。方法:选取2004年10月~2005年4月在本中心完成的42例患者42个周期为研究对象,前瞻性比较了两种精子分离方法的受精率、卵裂率、优质胚胎率、临床妊娠率、精子畸形率、精子回吸收率等。结果:两种方法分离精子所获得的ICSI胚胎移植(ICSI-ET)中,受精率、卵裂率、优胚率、临床妊娠率均无明显差异,但是改良上游法所获得的精子畸形率明显高于密度梯度离心法(P<0.01);重度少精子症密度梯度离心法回吸收精子优于改良上游法(P<0.01)。结论:在辅助生育技术ICSI-ET中,密度梯度离心法分离活动精子临床妊娠结局与改良上游法无明显差异;除对重度少精子症者外,均可以采用改良上游法。     

Less NO production and better motion parameter in human sperm by swim-up processing

Fifteen semen specimens were obtained from men for semen analysis; each was divided into two aliquots for prepararation. The motile sperm recovery rate, percentage motility, and motion parameters were measured for each semen specimen (n = 15) before and after preparation with the use of the two methods, and cultured with different time courses (1 hr, 3 hr, and 6 hr). Nitric oxide (NO) was measured using the chemiluminscence method after centrifugation. Recovery rate of motile cell was significantly higher in direct swim-up method (69.5 +/- 42.4% versus 49.3 +/- 29.3%, p < 0.05). In motility, direct swim-up method in the different time courses was significantly better than IxaPrep method. (1 hr: 91.1 +/- 5.2% vs 65.6 +/- 16.4%, 3 hr: 87.2 +/- 7.9% vs 65.2 +/- 16.5%, 6 hr: 86.1 +/- 7.5% vs 60.8 +/- 17.6% and prewash: 61.6 +/- 16.2%, p < 0.05). In VAP and VSL, the sperm prepared by the above two methods all improved compared to pre-wash sperm (p < 0.05), but there was no statistical significance between the two methods. NO production in the direct swim-up group was significantly lower than IxaPrep group in the first hour of culture (0.09 +/- 0.09 uM vs 0.15 +/- 0.09 uM, p < 0.05). NO production increased as the culture time increased in swim-up group, but conversed in IxaPrep group. The lower level of NO produced in the swim-up group may suggest that better sperm quality achieved is due to the decreased NO production.     

Sperm evaluation in cryopreserved bovine semen recovered by two selection methods

Previous experiments have established that various semen manipulation techniques are able to increase the qualitative features of the spermatozoa used in different techniques of assisted reproduction, but practically no comparative data on frozen-thawed bovine semen have been found. The aim of this study was to compare the efficacy of two sperm selection methods: centrifugation on Percoll gradient and filtration through a Sephadex ion-exchange column, to improve the recovery of motile and morphologically normal spermatozoa, without inducing sperm damage, from cryopreserved bovine semen samples. Semen samples were thawed and centrifuged on a discontinuous Percoll gradient, or were filtered through a Sephadex G-15-120 column with the addition of ion exchangers. Sperm concentration, percentages of motile spermatozoa, acrosome integrity, superoxide dismutase activity and lipid peroxidation were evaluated in recovered samples and controls. The motility of spermatozoa obtained by Sephadex ion-exchange filtration (88.87 +/- 6.37%) and by Percoll gradient centrifugation (83.00 +/- 6.21%) were significantly greater than that of control samples (60.14 +/- 8.44%). Other results disclosed that both sperm selection methods significantly increased the percentage of intact acrosome and superoxide dismutase activity. In both cases, the number of recovered spermatozoa diminished significantly versus untreated samples. Although the number of recovered spermatozoa was low, these methods were effective to select viable sperm from cryopreserved bovine semen.     

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.     

A new CentriSwim procedure to increase the recovery of motile spermatozoa

A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim-up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim-up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory-induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim-up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2-cm radius upward-directing arm, at an angle of 60 degrees for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22-24 degrees C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory-induced oligoasthenozoospermic specimens were significantly higher than following standard swim-up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim-up technique.     

Temporal decreases in sperm motility: which patients should have motility checked at both 1 and 2 hours after collection?

A decrease in sperm motility, and thus total motile sperm count (TMSC), over a period of hours might have clinical implications in counseling couples considering intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The objective of this study was to identify patients with decreases in sperm motility from 1 to 2 hours after collection and examine predictive relationships with semen analysis parameters. Between 2001 and 2005, 2313 semen samples were analyzed. Sperm motility was evaluated at both 1 and 2 hours after time of collection. Relevant seminal parameters were compared between patients, with a decrease in 1-hour to 2-hour motility (n = 384) compared with those that showed no change (n = 1929). The same analysis was performed in a subset of patients with a TMSC between 10 and 40 million. In the total patient population, only 16% (384/2313) demonstrated a decrease in 1-hour to 2-hour motility. In patients displaying a decrease in the 1-2-hour motility, sperm concentration (33.5 vs 79 million/mL, P < .0001) and percent normal morphology (7% vs 8%, P < .0001) were significantly lower. Additionally, a significantly higher incidence of 1-2-hour motility decrease was seen in patients with midpiece anomalies (33.3% vs 15.9%, P = .01). Within the subpopulation of 10-40 million TMSC, the only statistically significant difference was in patients with midpiece anomalies (80.0% vs 28.2%, P = .02) who demonstrated a higher incidence of the 1-2-hour motility decrease. Overall, patients with a TMSC between 10 and 40 million showed a significantly higher incidence of 1-2-hour motility decrease compared with the rest of the patient population (29.0% vs 14.6%, P < .0001). Because decreases in 1-2-hour sperm motility affect only a small portion of patients, it is not necessary to check 2-hour motility on all patients. However, because patients with a TMSC between 10 and 40 million were significantly more likely to show a decrease in sperm motility-a decrease that could have possible clinical implications in couples deciding between IUI, IVF, or ICSI--checking 2-hour sperm motility should be considered in this population.     

Swim-down separation of progressively motile sperm from poor quality human semen by the modified funnel procedure

The simultaneous swimming up and down from identical semen specimens suggested that the separation efficiency of progressively motile sperm by the swim-down method was superior to that of the swim-up method. The swimming down was performed in a small plastic funnel, the bottom of which was plugged with a 2.5 ml disposable syringe. In this funnel, 2.0 ml of 70% Percoll was poured and then the sperm resuspension was overlaid. After 1 h, progressively motile sperm penetrating into the lower part of Percoll layer (1.6 ml) was collected in the syringe. The technical strategies to yield higher density of progressively motile sperm were found to be centrifugal concentration of the sperm from whole ejaculate prior to the swimming down, and subsequent re-centrifugation of the separated sperm after the swimming down. Oligo-asthenozoospermic semen (22 +/- 4.4 x 10(6) ml1, 5.5 +/- 4.4% motility, n = 8) was processed by the present method, yielding sperm qualities of 32 +/- 19 x 10(6) ml1, 74 +/- 14% in the final preparations. Overall improvement in fertility index (sperm density ml1 x motility % x 10(8)) reached 27-folds on average.     

精液体外处理对精子核DNA链完整性影响的研究

目的 研究3种不同精子优选技术对精子运动参数及DNA链完整性的影响。方法 通过计算机辅助精液分析及彗星试验从精子运动参数及DNA链完整性两个方面评价精液体外处理对精子的影响。结果 上游法与Percoll密度梯度离心法相比,除前者精子回收率(15.499.39)%明显低于后者(42.807.17)%外,P<0.05,其他差别无显著性,PureSperm密度梯度离心法与Percoll密度梯度离心法比较,各运动参数差别无显著性(P>0.05)。精液经Percoll法及PureSperm法密度梯度离心和上游法处理后,总彗星细胞率较处理前降低(P<0.05)。结论 上游法、密度梯度离心法和Puresperm 均可以不同程度优化精液质量;对精子DNA链的损伤程度不同。     

Semen characteristics of the captive Indian leopard, Panthera pardus

Semen samples from 11 Indian leopards (Pantherapardus) from 3 different zoos in India were collected by electroejaculation. A computer-aided semen analyzer (CASA) was used for assessing the quality of the semen vis-à-vis sperm motility. The volume of the ejaculate, sperm density, and the number of motile and morphologically normal spermatozoa were found to be 1.57 +/- 1.26 mL, 55.78 million +/- 38.67 million per mL, 57.05% +/- 16.96% and 71.92% +/- 15.32%, respectively. Although the spermatology varied between individuals in the study, Box-Whisker-plot analysis suggested that the distribution was normal (P > .05). The ejaculated sperm were cryopreserved after diluting in test-yolk buffer. The post-thaw motility was 32.14% and did not differ at 30 or 60 days after cryopreservation. CASA indicated that the progressive velocity (VSL) of cryopreserved spermatozoa was decreased and, as a consequence, they moved more slowly than the neat (VSL 76.3 microm/sec in neat vs 53.8 microm/sec in cryopreserved spermatozoa) and the trajectories were less planar. However, both cryopreserved and neat spermatozoa penetrated the zona-free hamster oocyte with equal efficiency (79% neat vs 80% cryopreserved). The study also reports application of CASA for feline spermatozoa and provides information for the first time on the spermatology of the Indian leopard. This baseline data could be used in captive breeding programs. The results are compared and discussed with the available information on other felines.     

Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival.

Human semen was cryopreserved using Human Sperm Preservation Medium, TEST-Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a significantly better recovery of all semen parameters (motility, velocity, and recovery) than either TEST-Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences between donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen-to-specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and measurement variability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreservative and post-thaw motile count. This relationship allows the prediction of poor-thaw survival before freezing a specimen.     

Viability and fecundity of human semen specimens cryostored and transported at 5 degrees C using the Bio-Tranz shipping

This study was designed to assess the viability and fecundity of semen stored at 5 degrees C for 24 hours using the Bio-Tranz shipping system. Semen specimens were assessed for motility and sperm membrane integrity at the time of collection and 24 hours after storage in the Bio-Tranz. In group 1 (n = 61), specimens were diluted in TYB, processed and used for intrauterine insemination (IUI), leaving an aliquot for storage for 24 hours in the Bio-Tranz. In group 2 (n = 67), specimens were diluted in TYB, stored for 24 hours in the Bio-Tranz and then processed and used for IUI. In both groups, the total motile sperm used for IUI was similar and the women that underwent IUI were standardized for ovulation prediction and time of insemination. The overall sperm characteristics between the two groups were within normal range. Significant decreases were noted in sperm motility and membrane integrity in both groups after storage. Similar pregnancy rates were obtained between the two patient populations. The use of the Bio-Tranz shipper is extremely convenient for patients requiring semen evaluation, cryostorage or IUI and other assisted reproductive technologies.     

Sperm morphology and chromatin condensation before and after semen processing

Semen analysis constitutes the most important investigation of male infertility. However, the true anomalies present in defective sperm cells have been only partially characterized. The integrity of the sperm chromatin may play the most important role, particularly in ICSI, where most of the natural selection mechanisms are bypassed. This study was carried out to characterize sperm morphology (strict criteria), to evaluate chromatin condensation and sperm count in native semen as well as after semen preparation by the swim-up technique, and to eventually evaluate any correlation between these parameters. Semen from 90 men was analyzed for the above parameters in both the fresh and processed semen. Whereas the sperm count decreased after sperm preparation by the swim-up technique in comparison to the value in the fresh semen (p < .001), there was an increase in the percentage of morphologically normal (p < .001) and chromatin-condensed sperm (p = .99). However, there was no correlation between sperm morphology, chromatin condensation, and sperm count either in the fresh or in the processed semen samples. These results suggest that sperm morphology, sperm count, and chromatin condensation are independent parameters that should be evaluated separately in the assessment of male fertility in an assisted reproduction program.     

Glass wool filter preparation of cryopreserved spermatozoa

Summary: Oligozoospermic and asthenozoospermic semen ejaculates, as well as cryopreserved sperm samples prepared by the wash and swim-up procedure often result in unsatisfactory sperm recovery rates. In this study the glass wool filter and the wash and swim-up preparation procedures were compared on the basis of their 'effective' (number of live sperm per millilitre) recovery rates. The glass wool filter procedure consistantly produced significantly ( P = 0.0002) higher viable sperm concentrations, making it the preferred method for the preparation of cryopreserved sperm to be used in assisted reproduction techniques. The use of this preparation procedure has also been shown to have no adverse affect on the fertilizing potential of human spermatozoa in our unit.     

术前精液分析作为精索内静脉结扎术后生育功能恢复指标的探讨

目的:探讨精索静脉曲张的男性不育患者术前的精液分析结果,作为预测精索内静脉结扎术后精子活动及生育功能恢复指标的可行性。方法:诊断为男性不育的107例精索静脉曲张患者,以精液自动分析仪进行精液分析,据其活动精子总数(TMSC)≥20×106、(5~20)×106、<5×106分为A(n=32)、B(n=36)、C(n=39)3组。行左侧或双侧精索内静脉高位结扎术,术后3个月开始随访,进行精液分析,并了解其妻怀孕情况。结果:107例患者术后TMSC较术前有明显增加,但A、B组TMSC的绝对增加值明显高于C组(P<0.05);术前A、B两组68例中,术后有56例(82.4%)TMSC≥20×106,而C组39例中,术后仅8例(20.5%)TMSC≥20×106;患者妻子怀孕情况随访到98例,其中有36例自然妊娠,A、B两组怀孕率(56.3%和42.4%)与C组(12.1%)比较均有明显差异(P<0.05)。结论:精索内静脉高位结扎术对于术前TMSC≥5×106的精索静脉曲张所致男性不育患者是极为有效的治疗方法,而对于重度少弱精子症(TMSC<5×106)患者精子质量改善情况不佳。