深低温保存兔角膜神经形态的观察

采用氯化金染色、乙醛酸诱发荧光（ＧＩＦ）和乙酰胆碱酯酶染色（ＡＣｈＥＳ）技术分别对兔深低温保存角膜中总体神经、肾上腺能和乙酰胆碱能神经形态进行系统观察。结果表明，深低温保存角膜的氯化金染色和ＡＣｈＥＳ神经形态与正常角膜神经相一致，但ＧＩＦ却为阴性。提示在光镜下，深低温保存角膜过程对角膜神经不造成形态损害。     

Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing

In this study, we compared the effects of repeated freezing/thawing of human sperm by our in-house method of rapid freezing with slow programmable freezing. Sperm samples from 11 normozoospermic subjects were processed through density gradients and divided into three aliquots: non-frozen, rapid freezing and slow programmable freezing. Sperm in the rapid freezing group had better motility and viability than those in the slow freezing group (P<0.01) after the first, second and third cycles of freezing/thawing, but there was no difference in morphology. In the second experiment, rapid freezing was repeated three times in 20 subjects. The samples from each thawing cycle were evaluated for DNA fragmentation using the alkaline comet assay. DNA fragmentation began to increase considerably after the second cycle of freezing/thawing, but to a level that was not clinically important. In the third experiment, rapid freezing was done repeatedly in 10 subjects, until no motile sperm were observed after thawing. The median number of repeated freezing/thawing that yielded no motile sperm was seven (range: 5–8, mean: 6.8). In conclusion, we demonstrated that repeated freezing/thawing of processed semen using our rapid freezing method gave better results than standard slow programmable freezing. This method can help maximize the usage of precious cryopreserved sperm samples in assisted reproduction technology.     

人类精液收集与处理技术改进

辅助生殖技术中精子的准备是极其重要的一个环节.采集并分离得到高活力的精子是保证受孕成功的关键步骤,而精液的冷冻贮存是延续生育力的重要手段之一.目前,精子采集方法,各种精子分离技术和冷冻方式已广泛被临床采用.本文主要就从精液样本的采集、精子优选技术以及精液的冷冻贮存方式进行探讨.     

低渗水肿钝性分离法在深板层角膜移植中的应用（附视频）

目的评价应用低渗水肿钝性分离法的深板层角膜移植术治疗部分角膜疾病的临床疗效。方法回顾性分析2007年11月-2010年5月期『日J在温州医学院附属眼视光医院接受深板层角膜移植术患者109例（109眼）临床资料。所有患者在移植术中均采用无菌注射用水和钝性器械辅助分离的方法,移植供体为甘油冷冻保存的角膜移植片。术后随访6个月～1年,观察角膜移植片愈合情况、末次随访最佳矫正视力,分析术中植床穿孔率、术后双前房现象、移植片排斥反应率、原发病复发、高眼压及青光眼等并发症情况。结果所有患者术后最佳矫正视力均较术前有所提高,达到或超过0．4者89眼,0．1～0．3者16眼,低于0．1者4眼。有4眼发生后弹力层微小穿孑L（3．67％）,术后双前房现象在1～2周内消退。角膜上皮延迟愈合2眼,原发病复发4眼（单纯疱疹病毒性角膜炎2眼、细菌性角膜炎1眼、真菌性角膜炎1眼）,一过性高眼压1眼,经治疗均得到良好控制。无内皮型排斥反应、继发性青光眼和自内障发生。结论低渗水肿钝性分离法在深板层角膜移植中对治疗凶角膜基质混浊而致盲的角膜疾病疗效确切,手术难度降低,并发症减少,故而更易推广。     

Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men

The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5.6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.     

p53 and mitogen‐activated protein kinase pathway protein profiles in fresh and frozen spermatozoa

Sperm or testicular tissue cryopreservation is performed in cases of male infertility as a treatment for the preservation of fertility. When these sperm cells are used in assisted reproductive techniques, fertilisation rates, developmental and implantation potential of embryos decrease and the abortion rates increase. In the present work, differences of both phosphorylation and expression levels of p53 and Mitogen‐activated protein kinases (MAPK) proteins were analysed in 61 individual sperm samples before and after cryopreservation. We observed that p53 protein residue at Ser 15 was phosphorylated after cryopreservation. Because MAPK pathway activations may be involved in p53 phosphorylation, MAPK/ERK, Stress‐activated protein kinases (SAPK)/JNK and p38MAPK proteins were also investigated. Analysis showed that p38MAPK phosphorylations increased significantly. However, ERK and JNK expressions and phosphorylations decreased, although the differences were not statistically significant. According to our results, it may be suggested that cryopreservation process activates p53 via p38 MAPK pathway that subsequently causes apoptosis, which may be related to sperm parameters.     

Impact of using a fast‐freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast‐freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s^?1 or 75 °C 7 s^?1). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20 min post‐thawing in the fast‐freezing technique than in the conventional one. Plasma membrane integrity was also greater (P < 0.05) in semen frozen with the fast‐freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast‐freezing technique was 76% (19/25), and did not differ (P > 0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep‐horn insemination maximises the use of equine semen. The fast‐freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.