【作者】 崔险峰；

【导师】 孙光；

【作者基本信息】 天津医科大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 无精子症在一般男性人群的发生率约为1%,而在存在不育问题的男性中约为10%。无精子症可分为梗阻性无精子症(OA)和非梗阻性无精子症(NOA)。睾丸活检显示NOA可表现为三种病理类型：生精功能低下、成熟阻滞和唯支持细胞综合征。在以往,NOA患者被认为是无法治愈的,只能通过领养或供精解决后代问题。在1993年Schoysman R等人从OA患者的睾丸中提取睾丸精子用于体外受精(IVF)和卵胞浆内单精子注射技术(ICSI)获得成功。1995年DevroeyP等人从NOA症患者的睾丸中提取精子并通过ICSI使卵子成功受精。从此,对NOA患者的治疗而言,关键是如何高效的通过外科取精方法获得足够多的、高质量的活动精子。为达此目的,先后出现了几种从NOA症患者的睾丸中提取精子的方法。这些方法包括如下：(一)睾丸精子抽吸术(Testicular sperm aspiration,TESA);(二)针吸活检取精(Needle aspiration biopsy, NAB);(三)睾丸活检穿刺针穿刺取精(Cutting needle biopsy, CNB);(四)睾丸切开活检取精(Testicular sperm extraction,TESE);(五)单一曲细精管活检术(Single seminiferous tubule biopsy,SSTB);(六)显微切开睾丸切开取精(Microdissection TESE);(七)B超引导下睾丸取精术(Ultrasound-guided testicular sperm extraction)。如何简单、高效的通过外科方法,从NOA患者的睾丸中获得精子?对NOA患者的临床治疗有重大的现实意义。由于每次从NOA患者的睾丸中获得的精子很少,对这些睾丸精子的冷冻保存,因其数量极少,且精子细胞膜尚未完全成熟,精子核DNA的稳定性也较差。采用常规精液标本那样的冷冻保存办法已被证明效果很差。近年来随着人们对玻璃化冷冻研究的不断深入,研究者逐渐认识到,人类的精子可以在不使用冷冻保护剂的情况下,通过一种称为玻璃化冷冻方法成功的进行冷冻和复苏。这种冷冻方法要求在冷冻和解冻时,要以很高的降温速度进行[达到(7.2×105)℃/min],以实现精子的玻璃化冷冻,在这一过程中,极高的冻融速率使得精子内外液之间几乎同步地由液态转为玻璃态(冷冻时),或由玻璃态转为液态(解冻时)并无细胞内外液间的交换,故精子内外不会有冰晶的形成,从而避免了精子内产生冰晶对精子的机械性损伤,也消除了精子外冰晶形成引起的理化损伤。现有的研究资料显示,玻璃化冷冻保存精子,方法简单、快速,而且不需专门的冷冻设备。研究和探索微量精子的玻璃化冷冻,对NOA患者的临床治疗亦有重大的现实意义。第一部分两种外科取精方法在造模少精子症或无精子症大鼠获精率的比较目的：比较单个曲细精管取精术和常规睾丸切开取精术在造模少精子症或无精子症大鼠中的获精率,以期为临床找到一种简单、微创和高效的针对非梗阻性无精子症的外科取精方法。方法：45只实验雄性大鼠分A、B、C、D、E、F、G、H和Ⅰ共九组。A组、B组和C组,每组9只大鼠；D组、E组、F组、G组、H组和Ⅰ组,每组3只大鼠。每只实验大鼠一次性腹腔注射白消安和环磷酰胺,剂量分别为5mg/kg和40mg/kg(A组),5mg/kg和80mg/kg(B组),5mg/kg和120mg/kg(C组),10mg/kg和40mg/kg(D组),10mg/kg和80mg/kg(E组),10mg/kg和120mmg/kg(F组),15mg/kg和40mg/kg(G组),15mg/kg和80mg/kg(H组),15mg/kg和120mg/kg(Ⅰ组)。注射后将九组大鼠混合后常规饲养。于注射后第15天、第20天、第25天和第30天,每次随机取7只大鼠用于实验；第35天将剩余的大鼠全部用于实验。在实验过程中共有10只大鼠死亡,共有35只大鼠最终接受了睾丸外科取精子术。对每只接受睾丸外科取术的大鼠,实验方法为同一大鼠的一侧睾丸采用单个曲细精管取精,另一侧睾丸采用常规睾丸切开取精。两种取精方法所获得的睾丸组织处理后立即制成湿片,在倒置显微镜下放大200倍寻找精子。结果：45只实验大鼠,造模后10只死亡,最终进入实验性外科取精的大鼠35只。采用单个曲细精管活检取精方法共活检35只造模大鼠的一侧睾丸,获精率为65.7%(23/35)；采用常规睾丸切开活检取精方法活检其另一侧睾丸,获精率为40%(14/35)。第二部分采用不同的微载体和不同的冷冻方法对小鼠睾丸单个精子冷冻复苏效果的比较目的：比较采用不同的微载体和不同的冷冻方法对小鼠睾丸单个精子的冷冻复苏效果,以期为临床找到一种简单、高效的,针对非梗阻性无精子症患者睾丸微量精子的冷冻保存方法。方法：10周龄雄性昆明小鼠,通过睾丸切开活检取精法,获得睾丸组织,物理研碎后,经体外培养,单层密度梯度离心后获得小鼠的睾丸精子。A组采用透明带不加冷冻保护剂的玻璃化冷冻；B组采用ICSI针不加冷冻保护剂的玻璃化冷冻；C组采用透明带常规冷冻；D组采用ICSI针常规冷冻,每组各冷冻50个小鼠睾丸活动精子。比较精子复苏后的回收率和活动率两项指标。结果：A组解冻复苏后精子回收率为96.0%(48/50),精子活动率为89.6%(43/48)；B组解冻复苏后精子回收率为84.0%(42/50),精子活动率为95.2%(40/42)C组解冻复苏后精子回收率为98.0%(49/50),精子活动率为61.2%(30/49)D组解冻复苏后精子回收率为90.0%(45/50),精子活动率为68.9%(31/45)第三部分小鼠睾丸单个精子的玻璃化冷冻对卵胞质内单精子注射治疗结果的影响目的：了解不加冷冻保护剂的玻璃化法冷冻小鼠睾丸精子对卵胞质内单精子注射治疗结果的影响。方法：通过睾丸切开活检取精方法获得小鼠睾丸精子。玻璃化冷冻组为采用不加冷冻保护剂的玻璃化法冷冻复苏后的小鼠睾丸精子,新鲜组为来自小鼠睾丸的新鲜精子,两组精子均采用卵胞质内单精子注射方法使卵子受精,比较两组的2-细胞胚胎的受精率。结果：玻璃化冷冻组共注射48个成熟卵子,2-细胞胚胎37个,2-细胞胚率为77.1%；新鲜组共注射55个成熟卵子,2-细胞胚胎44个,2-细胞胚率为80.0%。结论：1.和常规睾丸切开取精子方法相比,单个曲细精管活检取精子方法获精率更高,而且操作简单对睾丸创伤小,值得在NOA患者的睾丸取精中推广。2.在对来自睾丸的单个精子进行冷冻保存时,采用空透明带作为微载体在解冻复苏后精子回收率方面优于ICSI针,但采用ICSI针在解冻复苏后可获得更高的精子活动率；采用不加冷冻保护剂的玻璃化冷冻优于常规冷冻。3.采用不加冷冻保护剂的玻璃化法冷冻保存微量睾丸精子是一种安全、经济、有效的冷冻保存方法,冷冻复苏后采用ICSI技术可取得与新鲜精子相似的受精率。更多还原

【Abstract】 Azoospermia, defined as the absence of spermatozoon in the ejaculate after the assessment of centrifuged semen on at least two occasions. Approximately1%of all men and10%of infertile men are affected by testicular failure as a result of azoospermia. Non-obstructive azoospermia(NOA) refers to absence of spermatozoa in semen analysis due to minimal or no production of fully developed spermatozoa in the testicles, non-obstructive azoospermia may manifest as three kinds of pathological types by testicular biopsy: hpyospermatogenesis, maturation arrest as well as Sertoli cell only syndrome.Patients with non-obstructive azoospermia were considered incurable in the past, the only method to have a baby is adoption or using donor sperm.Remarkably, Schoysman R, et al extract sperm from testes of patients with obstructive azoospermia in1993, and it was successfully applied in IVF and ICSI.In1995, Devroey P, et al extract sperm from testes of patients with non-obstructive azoospermia, and it was fertilized by ICSI.From now on, to the patients with non-obstructive azoospermia, the key is how to obtain enough and high quality sperm through surgical methods.For this purpose, it has appeared several methods to extract sperm from testes of patients with non-obstructive azoospermia.It can be summarized as seven methods as follow:Testicular spermaspiration (TESA), Needle aspiration biopsy(NAB), Cutting needle biopsy(CNB), Testicular sperm extraction(TESE), Single seminiferous tubule biopsy(SSTB), Microdissection TESE, Ultrasound-guided testicular sperm extraction.It has great realistic meaning for the treatment for how to obtain sperm from patients with non-obstructive azoospermia simply and efficiently through surgical method. Rare sperm can be obtained from testes of patients with non-obstructive azoospermia. For cryopreservation, stability of sperm nuclear DNA is poor because its quantity is few and sperm cell membrane has not yet been fully mature.It has been proven that the effect using conventional cryopreservation as common is very poor.Along with deep research of vitrification, researchers gradually realized that human sperm can be cryopreservated and recoveried by the method of cryoprotectant-free vitrification.It requires a high temperature and speed (7.2X 105)℃/min to cryopreservation and recorvery.In this process, the high freezing and thawing rate makes sperm liquid both inside and outside change from liquid to vitrification or from virtification to liquid almost synchronously, and there is no exchange between inside and outside. So there is no ice crystals arround sperm, and it avoids the sperm mechanical damage caused by ice crystals in sperm. At the same time, physical and chemical damage caused by ice crystals outside the sperm is eliminated.research data shows sperm vitrification is simple and fast, and there is no special refrigeration requirement.It has important practical significance for patients with non-obstructive azoospermia to research and explore sperm vitrification.Section I Compare the sperm retrieval male rate of conventional testicular sperm extraction and single seminiferous tubule biopsy in male rat model with oligoazoospermia/azoospermiaObjective:Compare the sperm retrieval rate of conventional testicular sperm extraction and single seminiferous tubule biopsy in male rat model with oligoazoospermia/azoospermia.Methods:Forty-five male rats (8weeks age) were divided into9groups(A group, B group,C group,D group,E group,F group, G group,H group and Igroup). Every group is9rats in A, B and C group and is3rats in D,E,F, G,H and I group. All of the45male rats were injected intraperitoneally and cyclophosphamide in abdominal cavity, and the dose was5mg/kg and40mg/kg(group A),5mg/kg and80mg/kg(group B),5mg/kg and120mg/kg(group C),10mg/kg and40mg/kg(group D),10mg/kg and80mg/kg(group E),10mg/kg and120mg/kg(group F),15mg/kg and40mg/kg(group G),15mg/kg and80mg/kg(group H),15mg/kg and120mg/kg(group I). At the15th,20th,25th and30th day after injection,7male rats were treated each time with surgical recovery of sperm. At the35th day, the remaining male rats were treated. One testis was treated by single seminiferous tubule biopsy and the other one by conventional testicular sperm extraction in every male rat. The testicular tissue from the two methods was immediately made into wet piece, and then magnified200times for looking for sperm using an inverted microscope.Results:10of the45male rats were died after injection, the remaining35were successfully included in the experiment. One testis of every male rats in the35were retrieved sperm with single seminiferous tubule biopsy and the retrieval sperm rate65.7%(23/35); the other testis were with conventional testicular sperm extraction and the retrieval sperm rate40.0%(14/35).Section Ⅱ Comparative Study on Single Spermatozoa Cryopreservation Using Two Micro-carriers and Methods of Cryopreservation in MiceObjectives:Through comparing the result of Cryopreservation and thawing of mice single spermatozoa with different micro-carriers and different freezing methods, to find more favorable freezing method for testicular spermatozoa from men with non-obstruction azoospermia.Methods:Obtain the testicular tissue by surgical biopsy of testis from a10-weeks old Kunming mice. And then got the testicular spermatozoa after physical pestle, cultured in vitro, and single density gradient centrifugation. A group is cryoprotectant-free vitrification with empty zona pellucida; B group is cryoprotectant-free vitrification with needle of ICSI; C group is conventional Cryopreservation with empty zona pellucida; D group is conventional Cryopreservation with needle of ICSI.Results:The recovery rate and the motility of spermatozoa in group A was96.0%(48/50) and89.6%(43/48); in group B was84.0%(42/50) and95.2%(40/42; in group C was98.0%(49/50) and61.2%(30/49); in group D was90.0%(45/50) and68.9%(31/45)Section III The effect on intracytoplasmic sperm injection with fresh and cryoproectant-free vitrification of individual testicular spermatozoa in miceObjectives:To observe the effects of Cryoproectant-free vitrification of individual testicular spermatozoa in Mice on intracytoplasmic sperm injection (ICSI).Methods:Obtain the testicular sperm from a mice by surgical biopsy. The sperm in vitrification freezing group was freezing and recovery by cryoprotectant-free vitrification, and in fresh group was testicular spermatozoa from a mice. Every sperm was used to fertilize an oocyte by ICSI, and then compare the two-cell embryos(MII) rate in two groups.Results:48oocytes were injected in vitrification freezing group and got37MII oocytes, the MII oocytes rate was77.1%(37/48);55oocytes were injected in fresh group and got44MII oocytes, the MII oocytes rate was80.0%(44/55).Conclusion:1. The retrieval sperm rate was higher in single seminiferous tubule biopsy compared with conventional testicular sperm extraction; and the relatively simple operation limits testicular damage. It is worth to spread in non-obstructive azoospermia patient.2. When freezing single testicular sperm, the recovery rate was higher with empty zona pellucida than needle of ICSI, but the latter had higher motility of spermatozoa; cryoprotectant-free vitrification was more favorable than conventional cryopreservation.3. When freezing single testicular spermatozoa, cryoprotectant-free vitrification was safety, economical and effective, and the fertility rate was similar to fresh sperm in ICSI.更多还原