浙江省人类精子库供精者精液参数及精子耐冻性变化分析

目的 了解供精者捐精过程中精液参数、精子耐冻性变化以及为人类精子库实验室质量控制提供部分依据;方法 13261份精液样本均来自2006年9月至2010年12月浙江省人类精子库的1513例合格的供精者,年龄20～42周岁,职业以学生为主,对所有精液标本进行精液常规分析;达到冷冻标准的精液标本进行冷冻及冷冻复苏后精液常规分析.结果 1513例供精者中,每次捐精均合格的为343例,占22.7%;每次捐精(至少连续3次)均不合格的为68例,占4.5%;捐赠精液至少一次不合格的为1102例,占72.8%.捐赠的13261份精液中,有10158份冷冻复苏后达到外供标准,占76.6%;不合格的捐赠精液为3103份,占23.4%,其中,未达到冷冻标准的为1565份,占所有捐赠精液的11.8%;冷冻复苏后未达到外供标准(不耐冻精液)为1538份,占所有捐赠精液的11.6%.结论 人类精子库供精者每次捐赠的精液不仅精液参数变化较大,其耐冻性变化也很大;所捐赠的精液不合格比例较高.     

不同程度弱精子症的精子蛋白质差异表达分析

目的研究不同程度弱精子症蛋白质表达差异,以期揭示蛋白质改变在弱精子症发生、进展中的作用.方法用iTRAQ标记以及二维高效液相色谱/串联质谱联用的方法研究人类正常精子以及轻、中、重度弱精子症患者的精子蛋白表达,通过相关软件分析数据,比较正常精子与不同程度弱精子症精子蛋白间的变化情况.结果本研究发现1073个蛋白质改变与弱精子症相关,而这些蛋白中,蛋白表达是以下调表达占主要形式的.通过Panther进行弱精子症中蛋白质组的蛋白质进行分类,结果发现这些升高蛋白主要涉及15个生物过程;降低的蛋白质中除了涉及上升表达的15个生物过程外,还涉及内环境稳态生物过程蛋白.这些生物过程中,与生殖过程密切相关的蛋白有26个,其中上调有10个,降低的蛋白为16个.结论精子蛋白表达是以下调表达占主要形式的,而这种情况可能是弱精子症发生的一个重要原因.在弱精子症的发生、发展中,多蛋白通过不同的生物学作用和途径交互影响,可能是最终影响精子的发生和活动力的重要原因.     

精子冷冻技术及其对精子蛋白的影响

精子低温冷冻是保持精子无限期存活的一项技术,能够帮助保存男性生育力。然而,冷冻和复苏会对精子产生损伤并影响其功能。损伤的机制涉及物理和化学方面,比如冰晶形成、渗透压改变、DNA破坏和氧化应激等。在这些机制的研究中,很多都涉及精子蛋白的变化,本文就精子冷冻技术的发展及冷冻对精子蛋白的影响作一综述。     

两种蛋白质对精子冷冻保护的影响

本文对蛋黄及白蛋白两种蛋白在精子冷冻保护中的作用进行了比较，结果显示：尽管两种蛋白质对精子冷冻复苏后的活动率的影响没有明显的差异性，但在Ａ２３１８７诱导，ＦＩＴＣ－ＰＳＡ染色的精子顶体反应测试中，蛋黄显示出相对较好的保护作用；在临床人工授精效果的观察中，使用蛋黄保护剂的精子呈现了较高的怀孕率。     

捐精者精子冷冻复苏率影响因素分析

目的:探讨季节、血型及精液参数等对捐精者精子冷冻复苏率的影响。方法:回顾性分析陕西省人类精子库捐精者4 088份精液标本,研究季节、血型、禁欲时间、精液量、精子形态、冷冻前精子活力及浓度对精子冷冻复苏率的影响。结果:捐精者精子冷冻复苏率随着精子浓度增高而增加,相关性分析提示精子浓度与冷冻复苏率呈正相关(r=0.247,P0.01)。而精子冷冻前活力和精子冷冻复苏率呈负相关(r=-0.262,P0.01)。禁欲第6天组的精子冷冻复苏率[(70.2±5.4)%]明显高于其他禁欲时间组(P0.01)。精子正常形态率20%组的精子冷冻复苏率[(71.4±5.1)%]要高于其他各组(P0.01)。A型血精子冷冻复苏率明显高于B型血[(69.1±4.8)%vs(69.8±4.7)%,P0.01];季节、精液量与精子冷冻复苏率之间无明显相关性(P0.05)。结论:捐精者的精子浓度、活力、形态及禁欲时间对于预测精子冷冻复苏率有一定的价值,而季节、血型、精液量与捐精者精子冷冻复苏率无明显相关性。     

两种蛋白质对精子冷冻保护的影响

本文对蛋黄及白蛋白两种蛋白质在精子冷冻保护中的作用进行了比较，结果显示：尽管两种蛋白质对精子冷冻复苏后的活动率的影响没有明显的差异性，但在Ａ２３１８７诱导，ＦＩＴＣ－ＰＳＡ染色的精子顶体反应测试中，蛋黄显示出相对较好的保护作用；在临床人工授精效果的观察中，使用蛋黄保护剂的精子呈现了较高的怀孕率。     

冷冻复温后低温保护剂和精子激活剂对精子功能影响的研究

本文对冷冻复温后精子的功能进行了研究观察，发现精液保护剂对冷冻复温后精子的存活有明显的保护作用，而单纯应用卵黄或甘油溶液却对精子功能存在不利的影响。体外处理精子时应用ＡＴＰ，肌苷对精子的功能无任何的改善。     

一例供精人工授精反复失败的精子蛋白质组学分析

目的分析1例供精人工授精(AID)反复失败捐献者精子的蛋白质组成差异,探讨精子蛋白质组学变化与妊娠结局的相关性。方法 1例连续9个AID周期失败的捐献者作为实验组;AID成功率在50%以上的3例捐献者作为对照组。收集两组精液样品,每位志愿者取3份精液,每次取精前禁欲3～7d,提取精子全蛋白,应用双向凝胶电泳分离精子蛋白,并行MALDI-TOF/TOF-MS质谱鉴定。结果实验组在分析区域内检测到432个蛋白点,对照组有460个蛋白点,两组中共有347个匹配蛋白点,匹配率达80.32%。检测到22个差异蛋白点,其中实验组有11个蛋白点高表达,8个蛋白点低表达,在对照组中3个蛋白点是缺失的。结论本研究利用蛋白质组学技术研究了1例AID反复失败供精者的精子蛋白质表达差异,发现22个差异蛋白点,其生物学意义有待进一步的研究。     

睾丸精子冷冻复苏对ICSI助孕结局的影响

目的探讨睾丸精子冷冻复苏对ICSI助孕结局的影响。方法回顾性分析2010年1月至2014年9月因梗阻性无精子症在本院采用睾丸精子行ICSI助孕的214个周期的临床资料,其中107个周期采用冷冻复苏的睾丸精子(冻精复苏组),另外107个周期采用新鲜睾丸精子(新鲜精子组)。比较两组中女方的一般资料、受精率、卵裂率、可利用胚胎数、优质胚胎率,以及临床妊娠率。结果本研究中,107个周期冻存睾丸精子复苏均获成功;冻精复苏组与新鲜精子组行ICSI助孕后的受精率[(76.91±18.24)%vs.(75.35±20.62)%]、卵裂率[(94.69±5.29)%vs.(95.37±4.48)%]、可利用胚胎数[(4.67±2.52)vs.(4.20±2.75)个]、优质胚胎率[(64.47±26.08)%vs.(60.34±27.39)%]及临床妊娠率(52.24%vs.50.63%)比较均无显著性差异(P0.05)。结论睾丸精子冷冻复苏对ICSI助孕结局并无显著影响。     

冷冻复温后低温保护剂和精子激活剂对精子功能影响的研究

本文对冷冻复温后精子的功能进行了研究观察，发现精液保护剂（甘油－卵黄－枸橼酸钠）对冷冻复温后精子的存活有明显的保护作用，而单纯应用卵黄或甘油溶液却对精子功能存在不利的影响。体外处理精子时应用ＡＴＰ、肌苷对精子的功能无任何的改善。两种浓度的苯甲酸钠咖啡因（１２ｍＭ和４ｍＭ）对冷冻复温后精子在不同时间内的存活和ＣＭ穿透力均有明显的提高作用     

Parameters associated with sperm quality prior to chemotherapy in lymphoma patients

Sperm quality in lymphoma patients may be reduced even prior to initiation of chemotherapy. The objective of this study was to examine the relationship between lymphoma prognostic factors and sperm quality prior to chemotherapy. A retrospective cohort study was conducted in a Hadassah Medical Center sperm bank and the Hematology department. The cohort included 101 Hodgkin's and 90 non-Hodgkin's lymphoma patients that underwent sperm cryopreservation before chemotherapy between 1998 and 2015. Known lymphoma prognostic factors were compared between patients with normal and impaired sperm parameters. The Prognostic Score Ratio (PSR), an index representing the number of negative lymphoma prognostic measures that found in a lymphoma patient, was additionally calculated and compared between the groups. Among the prognostic factors of lymphoma, the following factors were found to be associated with impaired sperm parameters—low albumin (p < 0.001) and haemoglobin (p < 0.001) levels, B symptoms (p = 0.021) and PSR (p < 0.001). Logistic regression showed significant association of albumin and haemoglobin with reduced sperm quality (OR = 2.7 and OR = 13.5, p < 0.05; respectively). To conclude, low albumin and haemoglobin levels are related to reduced sperm quality. The linkage between these prognostic factors and sperm quality may be related to a general inflammatory status.     

人类精子冷冻损伤及保护的研究进展

精子的冷冻保存已广泛应用于人类辅助生殖技术,然而,精子在超低温状态下会经历结构、功能的损伤,这种损伤是否会影响到辅助生殖技术的成功率,目前仍然没有一致的共识,世界各地的研究机构一直在致力于精子损伤机制的研究以及冷冻保护的探索,本文就精子冷冻损伤的不同类型以及功能的改变机制进行探讨,结合当前保护研究的最新进展作一综述。     

Need for sperm retrieval and cryopreservation at vasectomy reversal

PURPOSE: Controversy exists on whether to obtain sperm for cryopreservation routinely at vasectomy reversal. With recent improvements in in vitro fertilization with intracytoplasmic sperm injection, it is now possible to obtain a small amount of testicular tissue for cryopreservation in the event of reversal failure. However, to our knowledge no studies exist of who is most likely to benefit from this procedure. MATERIALS AND METHODS: We reviewed 84 consecutive vasectomy reversals performed by 1 surgeon (J. I. S.) between July 1996 and March 2000 with followup available for 77. We grouped cases by procedure as vasovasostomy, vasoepididymostomy and vasovasostomy with vasoepididymostomy as well as bilateral or unilateral. Sperm was retrieved at reversal in 15 of 46 vasovasostomy (none used), 11 of 18 vasoepididymostomy (3 used) and 13 of 20 vasovasostomy with vasoepididymostomy (none used) cases. RESULTS: The overall anastomotic patency rate after unilateral or bilateral vasovasostomy, unilateral vasovasostomy with contralateral vasoepididymostomy and unilateral or bilateral vasoepididymostomy was 96%, 83% and 57%, respectively. The natural pregnancy rate without in vitro fertilization was 57%, 50% and 14%, respectively. The most recent vasoepididymal anastomoses were performed by the Berger triangulation technique with a 78% patency and 25% pregnancy rate. Only 8% of men with banked sperm eventually used it for assisted reproductive techniques, in whom unilateral or bilateral vasoepididymostomy failed in all. CONCLUSIONS: We currently do not recommend routine sperm retrieval for cryopreservation in men who undergoing vasovasostomy. We encourage men who require bilateral vasoepididymostomy to bank sperm at reversal. In men who undergo vasovasostomy with vasoepididymostomy we base the decision on preoperative counseling and intraoperative findings.     

Reliable single sperm cryopreservation in Cell Sleepers for azoospermia management

Conventional sperm freezing methods perform best when freezing sperm samples containing at least hundreds of spermatozoa. In this severe male factor infertility case series, we examined the reproductive outcomes in 12 intracytoplasmic sperm injection cases where spermatozoa used were frozen in Cell Sleepers. Cell Sleepers are novel devices in which individual spermatozoa can be frozen in microdroplets. The case series included five men with obstructive azoospermia, six with nonobstructive azoospermia and one with cryptozoospermia, in whom microscopic sperm retrievals from testicular sperm extraction (TESE), micro‐TESE extracts and a centrifugation procedure resulted in less than 50 spermatozoa. A total of 304 microscopically retrieved spermatozoa were frozen in 20 Cell Sleepers using a rapid manual cryopreservation method. A total of 179 mature oocytes were injected with recovered thawed spermatozoa, resulting in a fertilisation rate of 65.9% (118 of 179), with no total fertilisation failures. In 10 cases, an embryo transfer was performed, three on day 3 and seven on day 5, resulting in a per cycle pregnancy rate of 58.3% (seven of 12). Four of the pregnancies have progressed past 20 gestation weeks. The recovery and use of spermatozoa that were frozen in Cell Sleepers was uncomplicated and effective and eliminated the need to perform any microscopic sperm retrieval procedures on the day of oocyte collection. Modification of the routine sperm cryopreservation methodology to include the use of Cell Sleepers increases the range of sperm samples that can be effectively cryopreserved, to include men with severe male factor fertility.     

Goat semen cryopreservation with rainbow trout seminal plasma supplemented lecithin-based extenders

The objective of this study was to determine the optimum concentrations of rainbow trout seminal plasma (RTS) supplemented extenders for goat semen quality at post-thaw and after incubation. Five sexually mature Saanen goat (Capra aegagrus hircus) were used for semen collection. Pooled semen was diluted with soybean lecithin-based extender without RTS (control) or supplemented with different concentrations of RTS (1%, 2%, 4% or 8%), at a final concentration of 150 × 10⁶ spermatozoon/ml. Sperm motility, plasma membrane functional integrity (HOST), damaged acrosome (PSA-FITC), mitochondrial activity (rhodamine123) and DNA integrity (TUNEL) were evaluated. Spermatological parameters were evaluated at post-thaw and after 6 hr incubation. RTS8 group preserved sperm motility, acrosomal integrity, plasma membrane functional integrity and mitochondrial function better than the control group (p < .05). The study demonstrated that RTS supplemented lecithin-based extenders have useful effects on goat spermatozoa. In addition, the results of the current study represented the positive effect of using 8% RTS supplemented extender.     

Seminal plasma proteins and their relationship with sperm motility and morphology in boars

The identification of biomarkers associated with seminal traits could aid in the selection of higher quality ejaculates and benefit the swine industry. The objective of this study was to identify boar seminal plasma proteins associated with sperm motility and morphology. Twenty ejaculates from fifteen adult boars from a commercial boar stud were used for this work. After routine semen collection and analysis, ejaculates were classified into two groups: high‐quality semen (HQS) and low‐quality semen (LQS), based on sperm motility and morphology. Semen samples were processed for seminal plasma separation and analysis by 2D SDS‐PAGE. Total and progressive sperm motility differed between groups (p < 0.001), as well sperm morphology (p < 0.05). The intensity of spots identified as Major seminal plasma PSP‐I (PSP‐I) and cathepsin B (CTSB) was higher in LQS as compared to HQS samples (p < 0.05). Also, PSP‐I was positively associated with major and sperm cauda defects. Sperm motility was negatively correlated with both PSP‐I and cathepsin B. We conclude that high concentrations of Major seminal plasma PSP‐I and cathepsin B in boar seminal plasma are associated with reduced total and progressive sperm motility and low sperm morphology and might be used as biomarkers for semen quality.     

Cryopiece,a novel carrier with faster cooling rate,high recovery rate and retrieval rate,for individual sperm cryopreservation

BackgroundCryopreservation of extremely few spermatozoa is still a major challenge for male fertility preservation. This study aims to evaluate the cooling rate, recovery rate, and retrieval rate, along with other parameters of spermatozoa that cryopreserved using Cryopiece, a novel carrier, for individual sperm cryopreservation.MethodsSemen samples from 60 fertile donors were collected, and each semen sample was screened for motile sperm and mixed with cryoprotective agent (CPA), and then frozen using Cryopiece, micro-straw, and mini-straws. The cooling rate, retrieval rate, and recovery rate, morphology, DNA fragmentation index (DFI) and mitochondrial membrane potential (MMP), were compared among the un-frozen sperm and the sperm cryopreserved using these carriers.ResultsCryopiece possessed the fastest cooling rate. After freeze-thaw, the average retrieval rate of sperm cryopreserved using Cryopiece was 96.25%, and the average recovery rate was 64.40%, which were higher than that of sperm cryopreserved using the other two carriers (71.42% and 54.30% for micro-straw, and 63.54% and 58.04% for mini-straw, respectively). There was no significant impact on DFI after sperm cryopreservation, and no significant difference in morphology between sperm cryopreserved using these carriers was observed. Though MMP of sperm changed significantly after cryopreservation, micro-straw maintained sperm MMP better than Cryopiece and mini-straw did, while no significant difference was observed in MMP between sperm cryopreserved using Cryopiece and mini-straw.ConclusionsCryopiece produced satisfying retrieval and recovery rates in sperm cryopreservation and should be an ideal carrier for cryopreservation of small number of sperm.     

Effect of sperm pooling with seminal plasma collected in breeding or nonbreeding season on Saanen goat sperm cryosurvival

The aim of this study was to determine the effects of both the removal of seminal plasma (SP) and the pre‐freezing addition of seminal plasma collected during the breeding or nonbreeding season on goat sperm survival after thawing. Semen samples were pooled. One aliquot of pooled semen was used as a control group. Four aliquots were then centrifuged, and the SP was removed in Group I, pipetted but not removed in Group II, removed and then pooled for animals collected in the breeding season in Group III and removed and pooled for animals collected in the nonbreeding season in Group IV. Group samples were frozen and then were assessed for rates of sperm motility, plasma membrane functional integrity hypo‐osmotic swelling test (HOST), defective acrosomes (FITC‐PSA), DNA fragmentation (TUNEL) and mitochondrial membrane damage (Rhodamine 123). The results showed that pre‐freezing addition of SP collected in breeding season maintained post‐thaw sperm characteristics at 0 hr better than SP removal group, but removing seminal plasma showed positive effects on spermatozoa, as incubation time increased to 5 hr. In conclusion, the pre‐freezing addition of seminal plasma did not maintain post‐thaw goat sperm characteristics as successfully as in the groups with seminal plasma removed after an incubation period.     

Phospholipid composition of human sperm and seminal plasma in relation to sperm fertility

The phospholipid and fatty acid composition of sperm was studied in 8 healthy and 16 infertile men. Infertile men randomly formed from the patients with normal semen parameters according to WHO criterion. Therefore, all semen parameters of infertile patients were similar to the same characteristics of the semen of healthy men, except the abnormal forms. The amount of abnormal forms in infertile men was significantly higher than in healthy men. Sperm from infertile men show a drastic loss of phosphatidyl ethanolamine. At the same time, the significant increase of phosphatidyl serine in the sperm and seminal plasma of sterile patients was found. Lysophosphatidyl serine in the sperm of the infertile men was detected. Fatty acid composition of the semen of infertile men was altered. The levels of stearic and n-3 polyunsaturated fatty acids (eicosopentaenoic and docosahexaenoic acids) was dramatically lowered, but the values of some n-6 polyunsaturated fatty acids (linolenic and docosatetraenoic) acids increased. There was significant positive correlation between docosahexaenoic acid and sperm motility (r = .82, p < .001) and negative correlation between linolenic acid and spermatozoa motility (r = -0.58. p < .05). Infertility of men with normal semen quality can originate from the disorder of sperm lipid metabolism. The drastic loss of phosphatidyl ethanolamine and n-3 polyunsaturated fatty acids with simultaneous enhancement of phosphatidyl serine and some n-6 polyunsaturated fatty acids in sperm could be an important cause of male infertility.