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活精子 不孕症是常见的;它影响了15%的总人口与730万美国夫妇 每年都要接受不孕症评估女性和男性都可能不育。最严重的 男性不育症是指在射精中找不到精子（10-20%的病例）;一种情况 也就是无精子症 精子是在睾丸中产生的。据信，大多数没有精子的男人， 射精后，睾丸中可能仍有极少量的精子在产生。我们有 开发了一种新的方法来检测和分离精液中的少量精子。在这 建议，我们将测试这一应用程序，以确定是否可用的精子可以找到显微手术， 目前的治疗方法已经失败。我们将首先比较酶处理睾丸 组织与睾丸活检的机械“切碎”，以测试这些组织制备 方法将导致精子与体细胞和生殖细胞更好的分离，从而产生更高的 可用精子的数量然后，我们将测试是否从睾丸活检中取出体细胞， 对样本进行分类，将增加从严重不育男性中回收的精子数量。 随后，我们还将使用选定的荧光染料组合测试分选后的精子， 对精子活力、运动性、DNA结构和线粒体功能有任何负面影响， 与未分类的精子相比。最后，我们将比较受精潜力，胚胎发育， 以及在动物模型中使用分选的和未分选的精子的妊娠率，以确保 使用的染料不会对生殖产生任何有害影响。 这些拟议的实验将提供必要的信息，开始在人类中进行临床试验， 我们可以分离活精子进行体外受精， 其中使用当前可用的治疗方法没有发现精子。

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.