Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw (LSL straw) for freezing few spermatozoa for intracytoplasmic sperm injection (ICSI). Semen samples from 22 fertile donors were collected, and each semen sample was diluted and mixed with cryoprotectant in a ratio of 1:1, and then frozen using three different straws such as LSL straw (50-100 μl), traditional 0.25 ml and 0.5 ml straws. For freezing, all straws were fumigated with liquid nitrogen, with temperature directly reducing to −130-−140°C. Sperm concentration, progressive motility, morphology, acrosome integrity, and DNA fragmentation index were evaluated before and after freezing. After freezing-thawing, LSL straw group had significantly higher percentage of sperm motility than traditional 0.25 ml and 0.5 ml straw groups (38.5% vs 27.4% and 25.6%, P < 0.003). Sperm motility and acrosomal integrity after freezing-thawing were significantly lower than that of before freezing. However, there was no significant difference in morphology, acrosome, and DNA integrity between the three types of straws (P > 0.05). As LSL straws were thinner and hold very small volume, the freezing rate of LSL straw was obviously faster than 0.25 ml straw and 0.5 ml straws. In conclusion, LSL micro-straws may be useful to store few motile spermatozoa with good recovery of motility for patients undergoing ICSI treatment.

Objective To evaluate the sperm retrieval rate (SRR) in non-obstructive azoospermia (NOA) associated with cryptorchidism and the effect of age at orchidopexy on SRR.Methods A total of 33 NOA after cryptorchidism (inguinal) and 46 NOA without cryptorchidism (as control) matched for testicular volume,follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels.The NOA after cryptorchidism underwent microdissection of testicular sperm extraction (MD-TESE) from September 2012 to March 2014.The SRR and testicular histology between the two groups were compared.The effect of age at orchidopexy (Group A:age 10 and younger; Group B:age older than 10) on testicular volume,hormone levels (FSH and LH) and SRR were analyzed.Results The SRR in NOA after cryptorchidism (70％,23/33) was significantly higher than that in control group (46％,21/46) (P＜0.05).The histology in NOA after cryptorchidism was significantly better than that in control (maturation arrest:64％ versus 39％ ; Sertoli cell-only syndrome:21％ versus 52％) (P＜0.05).No significant differences were found in testicular volume [(8.51±2.73) mlversus (8.80±5.74) ml],FSH [(24.83±7.63) U/Lversus (25.32±5.66) U/L],LH [(9.19±3.49) U/L versus (10.64±5.67) U/L] and SRR [73％ (11/15) versus 67％ (12/18)] between Group A and Group B (P＞0.05).Conclusions The SRR is significantly higher in NOA after cryptorchidism than that in NOA without cryptorchidism,and it might be the result of better histology in NOA associated with cryptorchidism.The age at orchidopexy shows no correlations with testicular volume,hormone levels and SRR in NOA after cryptorchidism.

OBJECTIVE:To investigate sperm function indexes that can be used to effectively evaluate the sperm donors' fertility so as to select healthy post-thaw semen samples and improve the success rate of assisted reproductive technology. METHODS:According to the pregnancy outcomes, we divided 40 donor semen samples into a high-fertility group (n = 20) and a low-fertility group (n = 20). We measured and compared the concentration, progressive motility, morphology, acrosome intactness, DNA integrity and mitochondrial membrane potential (MMP) of the post-thaw sperm between the two groups. RESULTS:There were statistically significant differences between the high- and low-fertility groups in the percentages of morphologically normal sperm ([18.50 +/- 6.10]% vs [14.42 +/- 6.44]%, P < 0.01), acrosome intactness ([86.17 +/- 4.49]% vs [80.04 +/- 7.52]%, P < 0.05) and DNA fragmentation index ([9.21 +/- 3.22]% vs [15.72 +/- 8.20]%, P < 0.05), but not in MMP ([56.75 +/- 18.80]% vs [52.23 +/- 18.86]%, P > 0.05). A significantly positive correlation was found between MMP and sperm motility (r = 0.760, P < 0.05), but not between other sperm functions and sperm concentration and motility. CONCLUSION:Sperm concentration, motility, morphology, acrosome intactness rate and DNA integrity contribute effectively to the evaluation of the fertilization capacity of post-thaw donor semen samples.

目的:优化电刺激取精术获取的精子处理和冷冻复苏方法,为临床不射精症的治疗和生育力保存提供新途径. 方法:将20例同房不射精患者电刺激获取的精液(或尿液)样本分为两组,其中6例采用PBS洗涤并用Modified-HTF重悬(A组),另外14例用Modified-HTF直接洗涤并重悬(B组),对处理后精子行CASA,分析精子浓度和活动率;采用麦管载体和一步熏蒸法冷活动率为(1.12±0.41)％,M-HTF处理组的精子活动率为(9.23±9.43)％;冷冻复苏后,A组精子复苏后活动率和冷冻复苏率分别为(0.24±0.41)％、8.37％;B组的精子活动率和复苏率显著增加,分别为(4.11±2.33)％、35.41％.同样,电刺激尿液样本中,PBS处理组的精子活动率为(1.30±0.51)％,M-HTF处理组的精子活动率提高至(5.48±5.32)％;冷冻复苏后,A组的精子活动率和复苏率分别为(0.26±0.40)％、6.31％;B组的精子活动率和复苏率,分别为(3.02±1.64)％、45.88％. 结论:本研究所建立的电刺激精子经体外培养及冷冻复苏的方法,可以显著增加精液和尿液样本中精子的活力,改善精子的冷冻复苏率,为临床不射精症的治疗和生育力保存提供新途径.

Objective To determine the correlation between semen parameters, sperm DNA damage, progressive motility (PR), morphology and intrauterine insemination (IUI)/intracytoplasmic sperm injection (ICSI) outcomes. Methods All the donors providing the samples in this study were recruited by Shanghai Human Sperm Bank. For IUI, 122 donors were divided into group A (n=60) and group B (n=62). Group A had a higher pregnancy rate while group B had a lower pregnancy rate (3.86 ± 1.50% vs 0.18 ± 0.52%). For ICSI, 45 donors were divided into group C with a higher pregnancy rate (77.78 ± 17.21%, n=23), group D with a lower pregnancy rate (40.73 ± 19.19%, n=22) and group E with an average pregnancy rate in the sperm bank (48.96 ± 12.08%, n=23). Semen analysis, morphology and DNA damage were assessed on samples retained in the sperm bank. Fresh semen samples were also collected and corresponding semen analyses data was included along with the pregnancy rates. Results No significant difference was found in the population characteristics between groups A and B, while there was a significant difference in sperm DNA fragmetation index (DFI) and morphology between the two groups (P 0.05). There was no significant difference in population characteristics between groups C, D and E while the DFI of group D was significant higher than groups C and E (P 0.05). Conclusion DFI might be a good predictor for IUI outcomes. Infertile couples with a high DFI should choose ICSI treatment instead of IUI. DFI should be a routine screening marker used to screen for sperm donors.

OBJECTIVE To analyze the distribution characteristics of the main semen parameters of healthy semen donors and normal fertile men in Shanghai, compare the semen quality between the two groups, and investigate the normal reference values of the semen parameters of the fertile population in Shanghai. METHODS We obtained semen samples from 100 healthy donors and 41 fertile men, performed semen analyses according to the WHO (2010) guidelines, and determined the semen volume, sperm concentration, sperm progressive motility, total sperm count and total progressively motile sperm count. We analyzed the distribution of the semen parameters of the normal fertile men, and obtained the lower limits of their normal reference values. RESULTS There were no statistically significant differences in the main semen parameters between the healthy donors and normal fertile men (P < 0.05). The lower reference limits for the semen parameters of normal fertile men in Shanghai (P < 0.05) were as follows: sperm concentration > or = 27.3 x 10(6)/ml, sperm progressive motility > or = 8.1%, semen volume > or = 0.82 ml, total sperm count > or = 44.73 x 10(6) per ejaculate, and total progressively motile sperm count > or = 24.68 x 10(6) per ejaculate. CONCLUSION For the evaluation of male fecundity, total sperm count and total progressively motile sperm count may be two better predictors than others.

OBJECTIVE:To investigate the ectopic grafts of mouse testicular cells by observing the reconstruction of seminiferous tubules, colonization of spermatogenic cells and spermatogenesis using immunodeficient mice as recipients.METHODS:The testes of newborn male ICR mice were digested to obtain single cell suspension. The cells were then mixed with matrigel and subcutaneously grafted into the dorsal region of the male nude mice. The mice were castrated after the operation and the grafts were dissected from 5 of the nude mice at 4, 6, 8 and 10 weeks, respectively. The success rates of transplantation and the graft diameters were calculated, and the structure of the reconstituted seminiferous tubules, colonization of the germ cells and spermatogenesis were observed by HE staining and immunohistochemistry.RESULTS:All the mice recipients survived after the testicular cell transplantation. Within 10 weeks after the operation, tissue masses could be observed, with the diameter increased from (3.91 +/- 0.71) mm at 4 weeks to (6.69 +/- 0.50) mm. Neovascularization was detected at the surface of the masses and seminiferous tubule structures found in the grafts. The germ cells that developed from spermatogonia to round spermatids were observed, but with no sperm in the tubules. Germ cells, Sertoli cells and Leydig cells were identified by immunochemical detection of Mvh, Gata4 and P450Scc in the grafts at 8 weeks.CONCLUSION:Seminiferous tubules could be ectopically reconstructed from suspension of neonatal mouse testicular cells. Ectopic grafting provided a preferable model for the studies on testis tissue engineering and interactions between testicular cells during testicular development and spermatogenesis.