首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
To compare the efficiency of sperm preparation between the two-layer Percoll gradient and mini-Percoll methods, 50 normal and 33 abnormal semen samples from male partners of infertile couples were studied. The number of recovered spermatozoa, percentage of motility, percentage of normal morphology, and their survival at 24 and 48 hours were assessed. Both Percoll gradient techniques resulted in a significantly higher percentage of motility and percentage of normal morphology compared with the original semen samples (p < 0.0001). The two-layer Percoll gradient showed a higher sperm recovery than the mini-Percoll method (p < 0.001), but the latter resulted in a higher percentage of motility (p > 0.001) and a higher sperm survival rate at 24 hours (p < 0.05) than the former, regarding normal semen samples. These differences did not appear with abnormal semen samples when analyzed as a group. Considering each of the abnormal parameters separately, sperm recovery was significantly higher after the two-layer Percoll gradient in the case of astheno- and teratozoospermia (p < 0.05), but sperm survival at 48 hours was higher after the mini-Percoll gradient in the case of teratozoospermia (p < 0.05). It is concluded that both the two-layer Percoll gradient and mini-Percoll method can be used effectively for sperm preparation. The former yields a higher sperm recovery, but the latter should be considered regarding teratozoospermic samples and semen samples of very low volume.  相似文献   

2.
OBJECTIVE: Sperm recovery using a single-layer Percoll procedure is significantly better than using the swim-up technique for infertile men and patients with normal sperm characteristics; however, in normal men results have been contradictory. Some studies have shown further improvement in semen quality with multiple layers. Therefore, this study compared the effect of single-layer and two-layer Percoll procedures on sperm characteristics of normozoospermic men. METHODS: Semen specimens from 10 normal donors were processed by layering 1 mL of the liquefied ejaculate on a single layer of 80% Percoll or on a two-layer (47% and 90%) Percoll gradient. Computer-assisted semen analysis was done to examine total motile sperm, percentage of recovery of motile cells, percent motility, curvilinear velocity, linearity, and amplitude of lateral head displacement. Each specimen was evaluated by the hypo-osmotic swelling (HOS) test, bovine cervical mucus penetration test, viability (eosin-nigrosin stain), and sperm morphology (World Health Organization and Kruger's strict criteria). RESULTS: Specimens processed with the two-layer Percoll procedure had significantly better recovery of spermatozoa, and significantly better percentage motility, linearity, amplitude of lateral head displacement, percentage tail swelling, and percentage viability than those separated on single-layer Percoll. Results for sperm morphology using WHO and Kruger's criteria were similar between the two methods (P = 0.92 for both sets of criteria). CONCLUSIONS: In normozoospermic men, the two-layer Percoll separation procedure significantly improves semen characteristics compared with separation on a single layer.  相似文献   

3.
OBJECTIVE: To compare the Enhance (Percoll; Conception Technologies, San Diego, CA) and PureSperm (Gen X International, Madison, CT) sperm preparation methods with respect to recovery (percentage of motile sperm), motility (%), path and progressive velocities (microm/s), and hyperactivation (%). DESIGN: Comparison of sperm processing methods. SETTING: University medical center-based clinical andrology laboratory and infertility program. PATIENT(S): Twenty-five men who presented for semen analysis. INTERVENTION(S): Each of 25 semen specimens were divided and each aliquot was prepared using two different density gradient centrifugation methods. MAIN OUTCOME MEASURE(S): The motile sperm recovery, percent motility, motion parameters, and percent hyperactivation were measured for each semen specimen (n=25) before and after separation with the use of the two methods. RESULT(S): There was no difference in the percent motility and motile count between specimens prepared with Enhance (Percoll) and PureSperm and fresh specimens. Statistically significant differences were found (fresh versus test) in the velocities and in hyperactivation (PureSperm only), and no differences were found between the processing methods. CONCLUSION(S): PureSperm appears to be as effective as Percoll (Enhance) for the recovery of good, progressively motile sperm for use in IUI or other assisted reproductive techniques.  相似文献   

4.
The aim of this study was to determine the relationship between calcium ionophore A23187-induced acrosome reaction (AR) and sperm fertilizing ability. Semen samples remaining after preparation for standard IVF were studied in 109 patients who had sperm concentrations > or =20 x 10(6)/ml. Ionophore-induced AR was performed on motile spermatozoa selected by centrifugation on a Percoll gradient. Semen analysis was performed using standard methods. Patients with higher (>50%, n = 76) fertilization rates had significantly higher ionophore-induced AR than patients with lower (<50%, n = 33) fertilization rates (49 +/- 14 versus 38 +/- 21%, P < 0.05). When the data from all patients were analysed by logistic regression, only the percentage sperm motility in insemination medium and ionophore-induced AR were significantly related to fertilization rates. Similar results were also obtained when the data from a subgroup of patients with poor (<15% normal) sperm morphology were analysed. However, when patients with normal sperm morphology > or =15% were analysed separately, only sperm count and the percentage of spermatozoa with progressive motility in semen were significantly related to fertilization rates. In conclusion, ionophore-induced AR was significantly related to fertilization rates in vitro mainly in patients with teratozoospermic semen. Tests for ionophore-induced AR may provide additional information about sperm fertilizing ability but may not indicate specific defects of the physiological AR.  相似文献   

5.
Oxygen radical generation is known to be detrimental to sperm function, especially motility, through the lipid peroxidation of the membranes. Generation of reactive oxygen species can be induced by leukocyte contamination, sperm centrifugation and the presence of abnormal spermatozoa with excess residual cytoplasm. This study aims to evaluate the effect on sperm motility of incubation in an antioxidant-containing solution, during liquefaction and centrifugation. Thirty semen samples were each divided into two equal parts: one mixed with Tyrode's solution, the other with a salt solution containing antioxidants (Sperm-Fit; Ellios Bio-Media, Paris, France). All the procedures were identical in the two groups. The ratio of leukocytes to spermatozoa was significantly correlated with the motility after liquefaction and after a 24 h incubation in routine in-vitro fertilization (IVF) medium and with the number of motile spermatozoa recovered after Percoll preparation. Moreover, when this ratio was > or = 0.2, all motility parameters were lowered. Incubation with Sperm-Fit allowed a higher percentage of motility after Percoll preparation when the ratio was > or = 0.2 (48 +/- 5% versus 41 +/- 6% for Sperm-Fit and Tyrode's solution respectively; P < 0.05) and a greater number of motile spermatozoa recovered after Percoll preparation, whatever the ratio (3.2 +/- 1.0 x 10(6) versus 2.4 +/- 0.7 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio > or = 0.2; 18.1 +/- 3.4 x 10(6) versus 14.4 +/- 2.9 x 10(6) for Sperm-Fit and Tyrode's solution respectively when ratio < 0.2; P < 0.05). These results show that incubation with antioxidants during liquefaction and centrifugation increases recovery of motile spermatozoa.  相似文献   

6.
The efficiency of the swim-up procedure and two-layer Percoll gradient centrifugation in procession of spermatozoa was assessed in ejaculates from 47 infertile men. A significantly higher total number of spermatozoa was harvested from Percoll gradients than from the swim-up procedure, the loss rates in concentration being -13.6 +/- 6.4% and -70.8 +/- 5.8%, respectively (p < 0.0001). Recovery in per cent motility was significantly higher after the Percoll gradient than after the swim-up procedure (34.8 +/- 10.2% versus -10.4 +/- 17.2%, p < 0.05). No significant difference was noted between the mean motility grades of the final solutions obtained by the two methods (2.7 +/- 0.2 and 2.0 +/- 0.4, respectively, p > 0.05). When evaluation was conducted within three initial fresh sample concentration categories such as severe oligospermia (lower than 5 x 10(6)/ml), moderate oligospermia (5 to 10 x 10(6)/ml) and mild oligospermia (higher than 10 x 10(6)/ml), the Percoll technique recovered significantly higher number of spermatozoa than the swim-up procedure through all concentration categories (p < 0.05 for each range). Despite being statistically insignificant, Percoll gradients produced final spermatozoa pools with higher per cent motility and motility quality within all concentration ranges. The results suggest that the Percoll gradient centrifugation should be the preferred selection method regardless of the initial fresh sample concentration.  相似文献   

7.
BACKGROUND: To determine if routine semen culture is useful in asymptomatic couples joining an in-vitro fertilization (IVF/ET) program. METHODS: Bacterial cultures and semen analysis according to WHO recommendations were performed on semen samples obtained before oocyte recovery from 88 asymptomatic couples undergoing IVF during a 7-month period. RESULTS: In 46 cultures at least one kind of microorganisms could be isolated. Forty-two cultures either contained bacterias regarded as normal skin flora (n = 14) or showed no growth of microorganisms (n = 28). No differences were found in sperm concentration, total sperm count and sperm morphology between the semen samples with positive bacteriology and those with negative culture results. Sperm motility was decreased before Percoll preparation if microorganisms were present. Positive culture results had no effect on either fertilization or pregnancy rates. CONCLUSIONS: These observations suggest that bacteriospermia is not associated with abnormal sperm function after Percoll preparation or adverse IVF outcome.  相似文献   

8.
The efficiency of spermatozoa prepared by swim-up or by Percoll centrifugation was assessed in an in-vitro fertilization programme on 71 semen samples of a well-defined quality [total number of type A (WHO criteria) motile spermatozoa]: category I (n = 21) with > 100 x 10(6), II (n = 31) with 15-100 x 10(6), III (n = 11) with 5-15 x 10(6) and IV (n = 8) with < 5 x 10(6) type A motile spermatozoa. Oocytes were inseminated 4 h after oocyte retrieval, alternately with spermatozoa derived from swim-up and Percoll preparation. Both selection procedures resulted in a significantly higher (P < 0.001) percentage motility as compared to fresh semen. For low-quality samples (III and IV), however, swim-up was more effective in selecting highly motile (P = 0.004) and morphologically normal spermatozoa (P < 0.05). For high-quality samples, this difference might have been masked by introducing a swim-up step to remove Percoll particles. Regardless of the initial sperm quality, the mean fertilization rate was significantly higher (P = 0.003) when Percoll-treated spermatozoa were used for insemination (51.3 versus 37.8%). For semen of groups I and II, no difference in fertilization capacity was observed according to the sperm preparation method. Despite the lower percentage motility and normal morphology for the Percoll compared to the swim-up treatment in groups III and IV, fertilizing capacity was significantly (P < 0.001) in favour of this selection method (65.3 versus 26.5% in group III, 47.6 versus 11.6% in group IV).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

9.
PURPOSE: Our purpose was to investigate whether a new, relatively hyperosmotic Percoll gradient, Enhance-S, can improve total motile sperm recovery rates compared with the commonly used Percoll gradient Perception. METHODS: Semen specimens from each of 17 donors were divided into two equal aliquots. One part was washed using Percoll Perception, while the other was prepared using Percoll Enhance-S. RESULTS: Compared to the unwashed specimen, sperm motion characteristics (motility and velocity) improved significantly after Percoll separation using either the Perception or the Enhance-S gradient. There was no difference in motility or velocity in spermatozoa recovered after wash with either of the two preparations. However, the total motile sperm recovery was significantly higher using the Percoll Enhance-S gradient than with the Percoll Perception gradient (P < 0.0024). CONCLUSION: The new Percoll Enhance-S gradient provides significantly more total motile sperm than the Percoll Perception gradient.  相似文献   

10.
OBJECTIVE: To look for correlations between acridine orange (AO) staining and semen parameters before and after sperm separation procedures and to assess whether the AO test predicts fertilization or pregnancy outcomes after standard IVF and intracytoplasmic sperm injection. DESIGN: Prospective study that simultaneously assesses sperm morphology and nuclear protein maturity on a cell-by-cell basis before and after preparative procedures. SETTING: University teaching hospital. PATIENT(S): Men (n = 140) undergoing diagnostic semen analysis. MAIN OUTCOME MEASURE(S): Acridine orange fluorescence of sperm nuclei, semen parameters, IVF outcome. RESULT(S): In unprocessed samples, 90% of sperm with normal heads displayed green fluorescence (mature nuclear protein); significantly lower percentages of green fluorescence were observed in sperm with abnormal heads. The percentage of mature normal sperm in the specimen correlated with motility. Sperm maturity after swim-up or Percoll gradient was significantly improved for sperm with normal or abnormal heads. The percentage of mature normal sperm correlated with motility after either Percoll or swim-up. Neither the percentages of mature nuclei nor mature normal nuclei correlated with fertilization or pregnancy outcome. CONCLUSION(S): Nuclear protein maturation correlates with sperm motility and morphology. Because morphologically normal and motile sperm are more mature, separation procedures should generate a population of sperm with the highest fertilization capacity. Acridine orange staining, however, did not predict fertilization efficiency or pregnancy outcome in IVF cycles.  相似文献   

11.
To compare the effectiveness of various sperm preparation methods, we examined sperm concentration, percentage of progressive motility, recovery rate, and various motion characteristics in 32 semen samples after Percoll and IxaPrep preparations. Except for sperm concentration, which was much higher after IxaPrep preparation, no statistical differences existed between these two methods in terms of motile sperm concentration (MSC), recovery rate, mean curvilinear velocity (VCL), mean straight-line velocity (VSL) and mean amplitude of lateral head displacement (ALH). Among 22 samples that were allocated in a 37 degrees C, 5% CO2 incubator, the mean MSC after 3 hours was significantly higher following IxaPrep preparation than following Percoll preparation (63.2 x 10(6)/mL vs. 42.8 x 10(6)/mL, p < .03). VCL and VSL were much higher after IxaPrep preparation than after Percoll preparation, both at 3 hours (p < .01) and at 24 hours (p < .03). ALH was also higher after IxaPrep preparation than after Percoll preparation, and it was statistically significant after 3 hours of incubation (p < .03). We conclude that the IxaPrep gradient may be adopted as the preferred method to the Percoll gradient in preparing spermatozoa for assisted reproduction because of the spermatozoa's persistent and better motion activities after incubation.  相似文献   

12.
The use of cryopreserved donor spermatozoa for insemination has become necessary to decrease the risk from sexually transmitted infectious diseases. Lower fecundity rates have been reported with this practice. Efforts have been applied to increase success, including identification of those sperm characteristics which correlate with increased fecundity. Data from in-vitro fertilization have revealed sperm morphology, motility and zona pellucida binding as important sperm parameters. Discontinuous Percoll gradient preparation yields a high concentration of motile spermatozoa. Using this preparation for thawed donor spermatozoa, we have identified post-preparation motility and progression as factors associated with increased fecundity. Consideration should be given to screening sperm donors with a freeze-thaw Percoll gradient preparation prior to acceptance into a donor bank.  相似文献   

13.
OBJECTIVES: To describe the experience of two male fertility programs using electroejaculation (EEJ) in the management of men with ejaculatory failure secondary to diabetes mellitus. METHODS: Twenty-nine EEJ procedures were performed in 7 diabetic men with ejaculatory failure. Results were reviewed with attention paid to sperm characteristics in both antegrade and retrograde specimens as well as pregnancy rates. RESULTS: Retrograde semen specimens retrieved from the bladder following EEJ contained a mean of 3444.5 million sperm (range 269.2 to 4996 million). Antegrade specimens contained a mean of 698.8 million sperm (range 226.8 to 1961 million). Mean sperm motility was 4% for retrograde specimens (range 0% to 11%) and 7% for antegrade specimens (1% to 15%). In all but 1 case, semen specimens were used for intrauterine insemination. The total number of motile sperm contained in the processed, inseminated specimens ranged from 1 to 87.2 million. In 1 case, the sperm obtained through EEJ was used in an in vitro fertilization procedure. CONCLUSIONS: EEJ can be successfully used to obtain sperm from men with ejaculatory failure due to diabetes mellitus. The procedure requires general anesthesia, and pregnancy rates after intrauterine insemination with the processed sperm are low. Advanced reproductive technologies may offer a feasible alternative, providing higher success rates with fewer procedures.  相似文献   

14.
We compared the efficacy of various methods of processing cryopreserved-thawed samples for the recovery of functionally adequate spermatozoa as assessed by the response to the sperm stress test (SST), an index of temperature activated sperm membrane lipid peroxidation, and immediate and delayed changes in sperm viability and motion parameters. Donor semen samples (n = 28) were cryopreserved-thawed and divided into six equal parts, one part was used as control and the remaining parts were used to compare five methods of sperm processing as follows: direct Percoll gradient processing, washing by one-step or stepwise addition of the washing medium followed by Percoll processing, and washing by one-step or stepwise addition of the washing medium. Additional samples (n = 10) were evaluated for the immediate and delayed (6 h at 37 degrees C) impact of one-step and stepwise washing (without Percoll separation). Compared with wash-only methods, samples processed using Percoll had a significantly higher SST score (P = 0.001), motility, rapid spermatozoa (>50 microm/s), curvilinear velocity and motility index (P < 0.001). Comparing various Percoll methods, direct Percoll processing resulted in the highest number of motile spermatozoa recovered (P < 0.00001) and a higher SST score based on curvilinear velocity (P = 0.001). Stepwise washing gave a significantly higher number of motile spermatozoa (P < 0.001) but with a significantly lower SST score based on the concentration of motile spermatozoa (P = 0.001), motility (P = 0.001) and motility index (P = 0.01). Sperm viability and motion parameters after 6 h of incubation showed no difference between one-step and stepwise washing. In conclusion, compared with wash-only methods, Percoll processed samples resulted in the recovery of spermatozoa with superior quality as assessed by SST and motion analysis. One-step washing of the samples gave an overall comparable recovery compared to the samples prepared stepwise. Having significantly higher SST scores, similar viability and the maintenance of motility, one-step washing may be a better method of processing thawed samples than the stepwise washing.  相似文献   

15.
OBJECTIVES: Although pre-freeze and post-thaw semen quality in patients with cancer is poor, it is not clear whether lipid peroxidation affects semen quality. This study assessed (1) whether poor semen quality in patients with cancer is caused by lipid peroxidation, and (2) whether patient age or sperm motility is associated with lipid peroxidation. METHODS: Lipid peroxidation was measured by determining malonaldehyde levels using the thiobarbituric acid method. Malonaldehyde levels were measured in cryopreserved semen specimens from patients with testicular (n = 15) or nontesticular cancer (n = 16) and normal men (control subjects, n = 20). A computer-assisted semen analyzer was used to determine the sperm concentration and motility before and after cryopreservation. RESULTS: Malonaldehyde levels did not differ in frozen-thawed semen specimens among patients with testicular (25.90 +/- 1.00 nM/10(8) sperm/hr) or nontesticular cancer (24.48 +/- 1.66 nM/10(8) sperm/hr), and control subjects (24.86 +/- 1.43 nM/10(8) sperm/hr). Malonaldehyde levels did not correlate with post-thaw sperm motility or patient age in patients with testicular or nontesticular cancer. Post-thaw sperm motility from patients with testicular or nontesticular cancer was significantly lower than that in normal subjects. CONCLUSIONS: The poor post-thaw semen quality in patients with testicular or nontesticular cancer is not related to lipid peroxidation but may be caused by other factors such as sperm membrane stress induced during the freeze-thaw process.  相似文献   

16.
OBJECTIVE: To determine whether the oral use of pentoxifylline improves the fertilization ability, concentration and motility of spermatozoa of poor fertilizer male subject. DESIGN: Analysis of the spermiogram parameters before and after oral pentoxifylline administration. SETTING: Zeynep Kamil Women and Children's Hospital, Department of Infertility. PATIENTS: Fourteen healthy, untreated male partners of couples who applied to our infertility department and were found to have poor semen parameters. MAIN OUTCOME MEASURES: Data of the semen analyses and fertilization rates are compared before and after treatment. RESULTS: Before the treatment, the average percentage sperm motility and density were 30.1 +/- 8 (%) and 14.8 +/- 3.2 (10(6)/ml), respectively. The same parameters were found to be 37.8 +/- 13 (%) and 19.5 +/- 10 (10(6)/ml), respectively, following the treatment. Not significant, p = 0.08 and p = 0.116, respectively). CONCLUSION: None of the semen parameters evaluated were improved by oral administration of pentoxifylline. Therefore pentoxifylline does not increase percentage motility and density of spermatozoa or the fertilization rates.  相似文献   

17.
Using three-colour fluorescence in-situ hybridization, sex chromosome ratios and frequencies of diploidy and disomy for chromosomes X, Y and 18 were compared in spermatozoa of good and poor motility after separation by swim-up, glass-wool and two-layer discontinuous Percoll methods. Semen samples were collected from seven normal males aged 26-31 years. A minimum of 6000 sperm nuclei per sample were evaluated for each chromosome for a total of 308,432 sperm nuclei. Hybridization efficiency was 99.8%. A slight change in the ratio of X- to Y-bearing spermatozoa was noted after Percoll separation (from 49.3:49.5 to 50.0:48.9; P = 0.036 and P = 0.046), but not after separation by the other two methods. We did not observe significant differences in the disomy rates for sex chromosomes or chromosome 18 or in the diploidy rate between spermatozoa with good and poor motility after separation by any of the three methods. Our data indicate that separation of motile spermatozoa does not alter the ratio of X- to Y-bearing spermatozoa to a degree that represents sex chromosome selection.  相似文献   

18.
PURPOSE: Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. METHODS: Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. RESULTS: Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. CONCLUSIONS: Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.  相似文献   

19.
OBJECTIVE: To determine whether type of cancer and response to treatment was related to prefreeze or post-thaw semen quality and to predict post-thaw sperm motility from prefreeze motility. DESIGN: Retrospective study. SETTING: Tertiary care institution. PATIENT(S): One hundred six cancer patients cryopreserving their semen specimens. INTERVENTION(S): Computer-assisted semen analysis was performed before and after cryopreservation on each patient specimen. MAIN OUTCOME MEASURE(S): The relationship of sperm motility and motion characteristics to type of cancer and patient's response to treatment. RESULT(S): Prefreeze and post-thaw semen quality did not differ between patients presenting with testicular cancer and Hodgkin's disease. Patients with leukemia or advanced soft tissue cancer had a higher prefreeze and post-thaw motility and higher total and motile sperm count than testicular and Hodgkin's disease patients. A prefreeze sperm motility of > or = 15% could predict a post-thaw motility of > 10%. CONCLUSION(S): Prefreeze or post-thaw semen quality in cancer patients is not affected (except the prefreeze motile sperm count within the testicular cancer patients) by the type of disease. Prefreeze motility can predict post-thaw motility. Cryopreservation of semen should be offered to cancer patients irrespective of the type of disease.  相似文献   

20.
OBJECTIVES: We evaluated the accuracy and precision of computer-assisted semen analysis (CASAs) to determine whether variations in the CASA results were related to the type of analyzer or to the type of specimens analyzed. METHODS: Semen specimens were analyzed manually and by CASA before freezing and after thawing. Multiple ejaculates (220 semen specimens) from normal healthy donors (n = 8) and 131 semen specimens from 57 patients with cancer were assessed. RESULTS: The differences between CASA and manual sperm counts and percent motility were higher in prefreeze and post-thaw specimens at sperm concentrations of less than 20 x 10(6)/mL. The differences between CASA and manual results were significant for cancer patients, compared with normal donors (P <0.01). CONCLUSIONS: CASA results are unreliable at sperm counts of less than 20 x 10(6)/mL and post-thaw motility is generally underestimated by CASAs.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号