| 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. |
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