Isolation of Viable Human Sperm from Failed Microsurgical Testicular Biopsies

项目摘要 从显微手术失败的睾丸活检中分离出可存活的人类精子 不孕症很常见；它影响着总人口的15%，美国有730万对夫妇 每年都要接受不孕不育的评估。女性和男性都可能不育。最严重的 男性不育的情况是在射精中找不到精子(10%-20%的情况)； 称为无精子症。 精子是在睾丸中产生的。据信，大多数没有精子的男性 精液中可能仍有极少量的精子在他们的睾丸中产生。我们有 开发了一种新的方法来检测和分离射精中的少量精子。在这 建议，我们将测试这一应用程序，以确定是否可以在哪里找到可用的精子，显微手术， 目前可用的治疗方法，已经失败了。我们将首先比较睾丸的酶处理方法 组织与机械“粉碎”的睾丸活检，以测试这些组织准备 这些方法将导致更好地从体细胞和生殖细胞中分离精子，从而产生更高的 可用精子数。然后我们将测试是否将体细胞从睾丸活检中移除，在 对样本进行分类，将增加从患有严重不育症的男性身上提取的精子数量。 随后，我们还将测试分离的精子是否使用选定的荧光染料组合， 对精子活力、活力、DNA结构和线粒体功能有任何负面影响，如 与未分选的精子相比。最后，我们将比较受精潜力，胚胎发育， 在动物模型中使用分类和未分类精子的妊娠率，以确保荧光 使用的染料，不会对生殖有任何不利影响。 这些拟议的实验将提供必要的信息，以启动人体临床试验，在 我们可以分离活精子进行体外受精，并对男性进行下游分析 在目前可用的治疗方法中没有发现精子。

Project Summary Isolation of Viable Human Sperm from Failed Microsurgical Testicular Biopsies Infertility is common; it affects 15% of the general population with 7.3 million American couples undergoing evaluation for infertility each year. Both women and men can be infertile. The most severe case of male infertility is where no sperm can be found in the ejaculate (10-20% of cases); a condition known as azoospermia. Sperm is produced in the testes. It is believed that the majority of men who do not have sperm in their ejaculate may have a minuscule number of sperm still being produced in their testicles. We have developed a novel methodology to detect and isolate a low number of sperm in the ejaculate. In this proposal, we will test this application to determine if usable sperm can be found where microsurgery, the current available treatment method, has failed. We will first compare the enzymatic processing of testis tissue with the mechanical "shredding" of a testis biopsy, to test which of these tissue preparation methods will result in a better separation of sperm from somatic and germ cells and thus yield a higher number of usable sperm. We will then test if removing the somatic cells from the testis biopsy, before sorting the specimen, will increase the number of sperm retrieved from men with severe infertility. Subsequently, we will also test if the sorted sperm, using the selected combination of fluorescent dyes, has any negative effect on sperm viability, motility, DNA structure, and mitochondrial function, as compared to unsorted sperm. Finally, we will compare the fertilization potential, embryo development, and pregnancy rates using sorted and unsorted sperm in an animal model, to ensure that the florescent dyes used, do not have any detrimental reproductive effects. These proposed experiments will provide the necessary information to start a clinical trial in humans, in which we can isolate live sperm for in vitro fertilization and utilize the downstream analysis for men where no sperm is found using the current available treatment methods.