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。
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/azoospermia
Objective: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 Mice
Objectives: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 mice
Objectives: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.
引文
1. Jarow JP, Espeland MA, Lipshultz LI. Evaluation of the azoospermic patient [J]. Urol,1989, 142(1):62-65.
2. Raman JD, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection is successful for the treatment of nonobstructive azoospermia associated with cryptorchidism [J]. Urol,2003,170(4):1287-1290
3. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia?A systematic review [J]. Hum Reprod Update,2007,13(6):539-549.
4. 杨军,刘继红,邹晓峰,等.非梗阻性无精子症患者的精子获得率及其预测指标—16年文献Meta分析[J].赣南医学院学报,2008,28(1):152-156.
5. Devroey P, Liu J, Nagy Z, et al. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia [J]. Human reproduction, 1995,10 (6):1457-1460.
6. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction [J]. Hum Reprod,1997,12(8):1688-1692.
7. Shah RS. Recent Advances in Infertility Management[In].Goenka ML, Goenka D, editors. Operative sperm retrieval for ART(C). Guwahati:Goenka,2001:179-183.
8. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia? A systematic review [J]. Hum Reprod Update,2007,13(6)-549.
9. Ishikawa T. Surgical recovery of sperm in non-obstructive azoospermia [J]. Asian Journal of Andrology,2012,14(1):109-115.
10. Fasouliotis SJ, Safran A, Porat-Katz A, et al. A high predictive value of the first testicular fine needle aspiration in patients with non-obstructive azoospermia for sperm recovery at the subsequent attempt [J]. Hum Reprod,2002,17(1):139-142.
11. Carpi A, Fabris FG, Todeschini G, et al. Large-needle percutaneous aspiration biopsy of the testicle in men with non-obstructive azoospermia [J]. Fertil Steril,2006,86(2):464-865.
12. Nowroozi MR, Ahmadi H, Ayati M,et al. Testicular fine-needle aspiration versus testicular open biopsy: Comparable sperm retrieval rate in selected patients [J]. Indian J Urol,2012,28 (1):37-42.
13. Shefi S, Kaplan K, Turek PJ.Analysis of spermatogenesis in non-obstructive azoospermic and virtually azoospermic men with known testicular pathology [J]. Reproductive BioMedicine Online,2009,18(4):460-464.
14. Carpi A, Sabanegh E, Mechanick J. Controversies in the management of nonobstructive azoospermia [J]. Fertil Steril,2009,91(4):963-970.
15. Beliveau ME, Turek PJ. The value of testicular'mapping'in men with non-obstructive azoospermia [J]. Asian J Androl,2011,13(2):225-230.
16. Esteves SC, Miyaoka R, Agarwal A. Sperm retrieval techniques for assisted reproduction [J]. Int Braz J Urol,2011,37(5):570-583.
17. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction [J]. Human Reprod,1997,12(8):1688-1692.
18. Ghalayini IF, Al-Ghazo MA, Hani OB, et al. Clinical Comparison of Conventional Testicular Sperm Extraction and Microdissection Techniques for Non-Obstructive Azoospermia [J].Clin Med Res,2011,3(3):124-131.
19. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia?A systematic review [J]. Hum Reprod Update,2007,13(6):539-549.
20. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision [J]. Hum Reprod,1999,14(1):131-135.
21. Tsujimura A, Matsumiya K, Miyagawa Y, et al. Conventional multiple or microdissection testicular sperm extraction a comparative study [J]. Hum Reprod,2002,17(11):2924-2929.
22. Amer M, Zohdy W, Abd El Naser T, et al. Single tubule biopsy:a new objective microsurgical advancement for testicular sperm retrieval in patients with nonobstructive azoospermia [J]. Fertil Steril,2008,89(3):592-596.
23. Ghalayini IF, Al-Ghazo MA, Hani OB, et al. Clinical comparison of conventional testicular sperm extraction and microdissection techniques for non-obstructive azoospermia [J]. Clin Med Res,2011,3(3):124-131.
24. Turunc T, Gul U, Haydardedeoglu B, et al. Conventional testicular sperm extraction combined with the microdissection technique in non-obstructive azoospermic patients:a prospective comparative study [J]. Fertil Steril,2010,94(6):2157-2160.
25. Ishikawa T, Nose R, Yamaguchi K, et al. Learning curves of microdissection testicular sperm extraction for non-bstructive azoospermia [J]. Fertil Steril,2010,94(3):1008-1011.
26. Ghalayini IF, Al-Ghazo MA, Hani OB, et al. Clinical comparison of conventional testicular sperm extraction and microdissection techniques for non-obstructive azoospermia [J]. Clin Med Res,2011,3(3):124-131.
27. Ando M, Yamaguchi K, Chiba K, et al. Outcome of microdissection testicular sperm extraction in azoospermic patients with Klinefelter syndrome and other sex-chromosomal anomalies [J]. Syst Biol Reprod Med,2013,13(1):21-25..
28. Dabaja AA, Schlegel PN. Microdissection testicular sperm extraction: an update [J]. Asian J Andrology,2013,15(1):35-39.
29. Shah RS. Recent Advances in Infertility Management[In].Goenka ML, Goenka D, editors. Operative sperm retrieval for ART [C]. Guwahati:Goenka,2001:179-183.
30. Amer M, Zohdy W, Abd El Naser T, et al. Single tubule biopsy: a new objective microsurgical advancement for testicular sperm retrieval in patients with nonobstructive azoospermia [J]. Fertil.Steril,2008,89(3):592-596.
31. Ramasamy R, Sterling'J, Fisher ES,et al. Identification of Spermatogenesis With Multiphoton Microscopy:An Evaluation in a Rodent Model [J].Urol,2011,186(6):2487-2492.
32. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia?A systematic review [J]. Hum Reprod Update,2007,13(6):539-549.
33. Hewitson L, Martinovich C, Simerly C, et al. Rhesus offspring produced by intracytoplasmic injection of testicular sperm and elongated spermatids [J]. Fertil Steril,2002,77(4):794-801.
34. Cohen J, Garrisi GJ, Congedo-Ferrara TA, et al. Cryopreservation of single human spermatozoa [J]. Hum. Reprod,1997,12(5):994-1001.
35. Abdel Raheem A, Rushwan N, Garaffa G, et al. Factors influencing intracytoplasmic sperm injection (ICSI) outcome in men with azoospermia [J]. BJU Int,2013,Jan 29[Epub ahead of print].
36. AbdelHafez F, Bedaiwy M, El-Nashar SA, et al. Techniques for cryopreservation of individual or small numbers of human spermatozoa:a systematic review [J]. Hum Reprod Update,2009,15(2):153-164.
37. Isachenko V, Isachenko E, Montag M, et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa [J]. Reprod Biomed OnIine,2005,10(3):350-354.
38. Isachenko E, Isachenko V, Weiss JW, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction,2008,136(2):167-173.
39. Nawroth F, Isachenko V, Dessole S, et al. Vitrification of human spermatozoa without cryoprotectants [J]. Fertil Steril,2002,23(2):93-102.
40. Isachenko V, Maettner R, Petrunkina AM, et al. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology [J]. Clin Lab,2011,57(9): 643-650.
41. Isachenko V, Maettner R, Petrunkina AM, et al. Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants:New Capillary Technology [J]. J Androl,2012,33(3): 462-468.
42. Satirapod C, Treetampinich C, Weerakiet S, et.al. Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing method:on motility, morphology, vitality and DNA integrity [J]. Andrologia,2012,44 (1):786-790.
43. Isachenko V, Isachenko E, Petrunkina AM, et al. Human spermatozoa vitrified in the absenc e of permeable cryoprotectants:birth of two healthy babies [J]. Reprod Fertil,2012,24(2):323-326.
44. Endo Y, Fujii Y, Kurotsuchi S,et al. Successful delivery derived from vitrified-warmed spermatozoa from a patient with nonobstructive azoospermia [J]. Fertil Steril,2012,98(6): 1423-1427.
45.崔险峰,王毅娜,张云山,等.不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏效果的比较[J].中国男科学杂志,2007,21(11):46-48.
46. Hsieh Y, Tsai H, Chang C, et al. Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae [J]. Fertil. Steril,2000,73(4):694-698.
47. Levi-Setti PE, Albani E, Negri L, et al. Cryopreservation of a small number of spermatozoa in yolk-filled human zonae pellucidae [J]. Arch Ital Urol Androl,2003,75(4):195-198.
48. Borini A, Sereni E, Bonu, et al. Freezing a few testicular spermatozoa reteieved by TESA [J]. Mol Cell Endorcrinol,2000,169(1-2):27-32.
49. Ye Y, Xu C, Qian Y, et al. Evaluation of human sperm function after being cryopreserved within the zona pellucida [J].Fertil Steril,2009,92(3):1002-1008.
50. Desail NN, Blackmon H, Goldfarb J. Single sperm cryopreservation on cryoloops:an alternative to hamster zona for freezing individual spermatozoa [J]. Reprod Biomed Online, 2004,9(1):47-53.
51. Isachenko E, Isachenko V, Weiss JW, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose [J]. Reproduction,2008,136(2):167-173.
52.靳镭,郑家凤,刘群,等.微滴无保护剂玻璃化法冷冻附睾微量精子的研究[J].中华男科学,2010,16(12):1089-1094.
53. Peng QP, Cao SF, Lyu QF, et al. A novel method for cryopreservation of individual human spermatozoa [J]. In Vitro Cell Dev Biol Anim,2011,47(8):565-572.
54. Endo Y, Fujii Y, Shintani K, et al. Simple vitrification for small numbers of human spermatozoa [J]. Reprod Biomed Online,2012,24(3):301-307.
55. Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia?A systematic review [J]. Hum Reprod Update,2007,13(6): 539-549.
56. Oehninge S. Clinical management of male infertility in assisted reproduction:ICSI and beyond [J]. Int J Androl,2011,34(5):319-329.
57. Cohen J, Garrisi GJ, Congedo-Ferrara TA, et al. Cryopreservation of single human spermatozoa [J]. Hum. Reprod,1997,12(5):994-1001.
58. AbdelHafez F, Bedaiwy M, El-Nashar SA, et al. Techniques for cryopreservation of individual or small numbers of human spermatozoa:a systematic review [J]. Hum Reprod Update,2009,15(2):153-164.
59. Isachenko V, Maettner R, Petrunkina AM, et al. Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants:New Capillary Technology [J]. Androl,2012,33(3): 462-468.
60. Isachenko E, Isachenko V, Weiss JW, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose [J]. Reproduction,2008,136(2):167-173.
61. Nawroth F, Isachenko V, Dessole S, et al. Vitrification of human spermatozoa without cryoprotectants [J]. Fertil Steril,2002,23(2):93-102.
62. Isachenko V, Maettner R, Petrunkina AM, et al. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology [J]. Cin Lab,2011,57(9): 643-650.
[1]Jarow JP, Espeland MA, Lipshultz LI. Evaluation of the azoospermic patient [J], J Urol, 1989,142(1):62-65.
[2]Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia?A systematic review[J]. Hum Reprod Update,2007,13(6): 539-549.
[3]杨军,刘继红,邹晓峰,等.非梗阻性无精子症患者的精子获得率及其预测指标—16年文献Meta分析[J].赣南医学院学报,2008,28(1):152-156.
[4]Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spennatozoon into an oocyte [J]. Lancet,1992,340(8810):17-18.
[5]Turek PJ, Ljung BM, Cha I, et al. Diagnostic findings from testis fine needle aspiration mapping in obstructed and nonobstructed azoospermic men [J]. J Urol,2000,163(6): 1709-1716.
[6]Schoysman R, Vanderzwalmen P, Nijs M, et al. Pregnancy after fertilization with human testicular spermatozoa [J]. Lancet,1993,342(8881):1237-1238.
[7]Devroey P, Liu J, Nagy Z, et al. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia [J]. Human reproduction, 1995,10 (6):1457-1460.
[8]Ghalayini IF, Al-Ghazo MA, Hani OB, et al. Clinical Comparison of Conventional Testicular Sperm Extraction and Microdissection Techniques for Non-Obstructive Azoospermia [J]. Clin Med Res,2011,3(3):124-31.
[9]Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia? A systematic review [J]. Hum Reprod Update,2007,13(6): 539-549.
[10]Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction [J]. J Urology,2005,65(6): 1190-1194.
[11]Okada H, Dobashi M, Yamazaki T, et al. Conventional versus microdissection testicular sperm extraction for nonobstructive azoospermia [J]. J Urol,2002,168(3):1063-1067.
[12]Amer M, Ateyah A, Hany R, et al. Prospective comparative study between microsurgical and conventional testicular sperm extraction in non-obstructive azoospermia:follow-up by serial ultrasound examinations [J].Hum Reprod,2000,15(3):653-656.
[13]Schill T, Bals-Pratsch M, Kiipker W, et al. Clinical and endocrine follow-up of patients after testicular sperm extraction [J]. Fertil Steril,2003,79(2):281-286.
[14]Turek PJ, Givens CR, Schriok ED, et al. Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with non-obstructive azoospermi [J]. Fertil Steril,1999,71(3):552-557.
[15]Nowroozi MR, Ahmadi H, Ayati M,et al. Testicular fine-needle aspiration versus testicular open biopsy:Comparable sperm retrieval rate in selected patient [J]. Indian J Urol, 2012,28(1):37-42.
[16]Carpi A, Sabanegh E, Mechanick J. Controversies in the management of nonobstructive azoospermia [J]. Fertil Steril,2009,91(4):963-970.
[17]Beliveau ME, Turek PJ. The value of testicular'mapping'in men with non-obstructive azoospermia [J]. Asian J Androl,2011,13(2):225-230.
[18]Esteves SC, Miyaoka R, Agarwal A. Sperm retrieval techniques for assisted reproduction [J]. Int Braz J Urol,2011,37(5):570-583.
[19]Shah RS. Advanced Infertility Management[In]. Hansotia M, Desai S, Parihar M, editors. Surgical and Non-Surgical Methods of Sperm Retrieval[C]. New Delhi:Jaypee Brothers, 2002:253-258.
[20]Amer M, Zohdy W, Abd El Naser T, et al. Single tubule biopsy: a new objective microsurgical advancement for testicular sperm retrieval in patients with nonobstructive azoospermia [J]. Fertil Steril,2008,89(3):592-596.
[21]Schlegel PN. Testicular sperm extraction:microdissection improves sperm yield with minimal tissue excision [J]. Hum Reprod,1999,14(1):131-135.
[22]Silber SJ. Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia [J]. Hum Reprod,2000,15(11):2278-2284.
[23]Amer M, Zohdy W, Abd E1 Naser T, et al. Single tubule biopsy: a new objective microsurgical advancement for testicular sperm retrieval in patients with nonobstructive azoospermia [J].Fertil Steril,2008,89(3):592-596.
[24]Ramasamy R, Schlegel PN. Microdissection testicular sperm extraction: Effect of prior biopsy on success of sperm retrieval [J]. J Urol,2007,177(4):1447-1449.
[25]Rasamamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction [J]. J Urology,2005,65(6): 1190-1194.
[26]Ghalayini IF, Al-Ghazo MA, Hani OB,et al. Clinical comparison of conventional testicular sperm extraction and microdissection techniques for non-obstructive azoospermia [J]. J Clin Med Res,2011,3(3):124-131.
[27]Schiff JD, Palermo GD, Veeck LL, et al. Success of testicular sperm extraction [corrected] and intracytoplasmic sperm injection in men with Klinefelter syndrome [J]. Clin Endocrinol Metab,2005,90(11):6263-6267.
[28]Turunc T, Gul U, Haydardedeoglu B, et al. Conventional testicular sperm extraction combined with the microdissection technique in non-obstructive azoospermic patients:a prospective comparative study [J]. Fertil Steril,2010,94(6):2157-2160.
[29]Ishikawa T, Nose R, Yamaguchi K, et al. Learning curves of microdissection testicular sperm extraction for non-bstructive azoospermia [J]. Fertil Steril,2010,94(3):1008-1011.
[30]Ghalayini IF, Al-Ghazo MA, Hani OB,et al. Clinical comparison of conventional testicular sperm extraction and microdissection techniques for non-obstructive azoospermia [J]. J Clin Med Res,2011,3(3):124-131.
[31]Ando M, Yamaguchi K, Chiba K,et al. Outcome of microdissection testicular sperm extraction in azoospermic patients with Klinefelter syndrome and other sex-chromosomal anomalies [J]. Syst Biol Reprod Med,2013,13(1):21-25.
[32]Dabaja AA, Schlegel PN. Microdissection testicular sperm extraction:an update [J]. Asian J Andrology,2013,15(1):35-39.
[33]Herwig R, Tosun K, Schuster A, et al. Tissue perfusion-controlled guided biopsies are essential for the outcome of testicular sperm extraction [J]. Fertil Steril,2007,87(5): 1071-1076.
[34]Esteves SC, Miyaoka R, Agarwal A. Spenn Retrieval Techniques for Assisted Reproduction [J]. Int Braz J Urol,2011,37(5):570-583.
[35]Bonarriba CR, Burgues JP, Vidana V, et al. Predictive Factors of Successful Sperm Retrieval in Azoospermia [J]. Actas Urol Esp,2012,21(4):47-51.
[36]Ramasamy R, Lin K, Gosden LV, et al. High serum FSH levels in men with nonobstructive azoospermia does not affect success of microdissection testicular sperm extraction [J].Fertil Steril,2009,92(2):590-593.
[37]Huang X, Bai Q, Yan LY,et al. Combination of serum inhibin B and follicle-stimulating hormone levels can not improve the diagnostic accuracy on testicular sperm extraction outcomes in Chinese non-obstructive azoospermic men [J]. Chin Med J,2012,125(16): 2885-2889.
[38]Reifsnyder JE, Ramasamy R, Husseini J,et al. Role of optimizing testosterone before microdissection testicular sperm extraction in men with nonobstructive azoospermia [J]. J Urol,2012,188(2):532-536.
[39]李敏,李凤华,杜晶,等.实时超声弹性成像评估无精子症睾丸生精功能的初步研究[J].中国超声医学杂志,2012,28(2):163-166.
[40]Carpi A, Sabanegh E, Mechanick J. Controversies in the management of nonobstructive azoospermia [J]. Fertil Steril,2009,91(4):963-970.
[41]Ramasamy R, Reifsnyder JE, Husseini J, et al. Localization of sperm during microdissection testicular sperm extraction in men with nonobstructive azoospermia[J]. J Urol,2013,189(2): 643-646.
[42]Stahl PJ, Masson P, Mielnik A, et al. A decade of experience emphasizes that testing for Y microdeletions is essential in American men with azoospermia and severe oligozoospermia [J]. Fertil Steril,2010,94(5):1753-1756.
[43]Schlegel PN. Nonobstructive azoospermia: a revolutionary surgical approach and results [J]. Semin Reprod Med,2009,27(2):165-170.
[44]Abdel Raheem A, Garaffa G, Rushwan N, et al. Testicular histopathology as a predictor of a positive sperm retrieval in men with non-obstructive azoospermia [J]. BJU Int,2013,111 (3): 492-499.
[45]Colpi GM, Colpi EM, Piediferro G, et al. Microsurgical TESE versus conventional TESE for ICSI in non-obstructive azoospermia:a randomized controlled study [J]. Reprod Biomed Online,2009,18(3):315-319.
[46]Abdel Raheem A, Garaffa G, Rushwan N, et al.Testicular histopathology as a predictor of a positive sperm retrieval in men with non-obstructive azoospermia [J]. BJU Int,2013,111(3): 492-499.
[47]丁攀,崔险峰,张云山,杨长海.非梗阻性无精子症患者外科取精技术的Meta分析[J].中国男科学杂志,2012,36(9):41-45.
[48]Achard V, Weber P, Mercier G, et al. High-magnification observation of seminiferous tubules through the tunica albuginea by two-photon laser scanning microscopy [J]. Asian J Androl,2011,13(5):774-776.
[49]Ramasamy R, Sterling J, Fisher ES, et al. Identification of Spermatogenesis With Multiphoton Microscopy:An Evaluation in a Rodent Model [J]. J Urol,2011,186(6):2487-2492.
[50]Ramkumar A, Lal A, Paduch DA, etal. Ultrasonically actuated silicon-microprobe-based testicular tubule metrology [J].Conf Proc IEEE Eng Med Biol Soc,2010,121(8):6469-6472.
[51]Greenhalgh JR, Griffith TS, Wald M. The Use of Immunofluorescence in Microdissection Testicular Sperm Extraction [J]. Androl,2009,30(5):548-551.
[52]Ostad M, Liotta D, Ye Z, et al. Testicular sperm extraction for non-obstuctive azoospermia:results of a multi-biopsy approach with optimized tissue dispersion [J].J Urol, 1998,52(4):692-696.
[53]Altay B, Hekimgil M, Cikili N,et al. Histopathological mapping of open testicular biopsies in patients with unobstructed azoospermia [J]. Br J Urol Int,2001,87(9):834-837.
[54]Donoso P, Tournaye H, Devroey P. Which is the best sperm retrieval technique for non-obstructive azoospermia.A systematic review? [J] Hum Reprod Update,2007,13(6):539-549.
[55]Turunc T, Gul U, Haydardedeoglu B, et al. Conventional testicular sperm extraction combined with the microdissection technique in nonobstructive azoospennic patients:a prospective comparative study [J]. Fertility Sterility,2010,94(6):2157-2160.
[56]Ben Rhouma K, Marrakchi H, Khouja H, et al. Outcome of intracytoplasmic injection of fresh and frozen-thawed testicular spermatozoa.A comparative study [J]. Reprod Med, 200,48(5):349-354.
[57]Abdel Raheem A, Rushwan N, Garaffa G, et al.Factors influencing intracytoplasmic sperm injection (ICSI) outcome in men with azoospermia [J]. BJU Int,2013,Jan 29[Epub ahead of print].
[58]Isachenko V, Isachenko E, Montag M, et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa [J]. Reprod Biomed Online,2005,10(3):350-354.
[59]Isachenko E, Isachenko V, Weiss JW, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose [J]. Reproduction,2008,136(2):167-173.
[60]Nawroth F, Isachenko V, Dessole S, et al. Successful cryopreservation of human spermatozoa by direct plunging into liquid nitrogen (vitrification) without cryoprotectants (abstract) [J]. Fertil Steril,2002,78(3):129.
[61]Isachenko V, Maettner R, Petrunkina AM, et al. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume:a novel technology [J]. Clin Lab,2011,57(9): 643-650.
[62]Isachenko V, Maettner R, Petrunkina AM, et al. Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants: New Capillary Technology [J]. J Androl,2012,33(3): 462-468.
[63]Satirapod C, Treetampinich C, Weerakiet S.et al. Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing method:on motility, morphology, vitality and DNA integrity [J]. Andrologia,2012,44(1):786-790.
[64]Isachenko V, Isachenko E, Petrunkina AM, et al. Human spermatozoa vitrified in the absenc e of permeable cryoprotectants:birth of two healthy babies [J]. Reprod Fertil,2012,24(2):323-326.
[65]Endo Y, Fujii Y, Kurotsuchi S,et al. Successful delivery derived from vitrified-warmed spermatozoa from a patient with nonobstructive azoospermia [J]. Fertil Steril,2012,98(6): 1423-1427.
[66]崔险峰,王毅娜,张云山,等.不采用冷冻保护剂的玻璃化法与常规冷冻法对人精子冷冻复苏效果的比较[J].中国男科学杂志,2007,21(11):46-48.
[67]Borini A, Sereni E, Bonu, et al. Freezing a few testicular spermatozoa reteieved by TESA [J]. Mol Cell Endorcrinol,2000,169(1-2):27-32.
[68]Ye Y, Xu C, Qian Y, et al. Evaluation of human sperm function after being cryopreserved within the zona pellucida [J]. Fertil Steril,2009,92(3):1002-1008.
[69]Desail NN, Blackmon H, Goldfarb J. Single sperm cryopreservation on cryoloops:an alternative to hamster zona for freezing individual spermatozoa [J]. Reprod Biomed Online, 2004,9(1):47-53.
[70]Isachenko E, Isachenko V, Weiss JW, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose [J]. Reproduction,2008,136(2):167-173.
[71]靳镭,郑家凤,刘群,等.微滴无保护剂玻璃化法冷冻附睾微量精子的研究[J].中华男科学,2010,16(12):1089-1094.
[72]Peng QP, Cao SF, Lyu QF, et al. A novel method for cryopreservation of individual human spermatozoa [J]. In Vitro Cell Dev Biol Anim,2011,47(8):565-572.
[73]Endo Y, Fujii Y, Shintani K, et al. Simple vitrification for small numbers of human spermatozoa [J]. Reprod Biomed Online,2012,24(3):301-307.