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Isolation of Viable Human Sperm from Failed Microsurgical Testicular Biopsies

从失败的显微外科睾丸活检中分离活的人类精子

基本信息

批准号：
9765342
负责人：
DARIUS A PADUCH
金额：
$ 8.48万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-16 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9765342
关键词：
Adopted Affect American Animal Model Animals Antibodies Apoptosis Apoptotic Area Biological Biological Assay Biology Biopsy Biopsy Specimen Birth Rate Cell Culture Techniques Cells Child Clinical Clinical Trials Couples DNA DNA Fragmentation DNA Structure Data Development Dyes Embryo Transfer Embryonic Development Ensure Enzymes Evaluation Fertilization Fertilization in Vitro Fluorescence Fluorescent Dyes General Population Germ Cells Human Impairment In Vitro Induction of Apoptosis Infertility Intracytoplasmic Sperm Injections Lasers Liquid substance Live Birth Male Infertility Measures Mechanics Methodology Methods Microsurgery Mitochondria Molecular Morphology Mus Nuclear Oocytes Operative Surgical Procedures Outcome Patients Pregnancy Rate Reaction Recovery Reporting Reproducibility Retrieval Rodent Running Safety Selection Bias Seminiferous tubule structure Sex Chromosomes Small RNA Somatic Cell Sorting - Cell Movement Specimen Sperm Count Procedure Spermatogenesis Stains Testicular Tissue Testing Testis Therapeutic Tissues Transgenic Mice Ultraviolet Rays Visible Radiation Woman Y Chromosome cell motility experimental group experimental study improved improved outcome leydig interstitial cell men mitochondrial dysfunction next generation sequencing novel reproductive risk minimization sertoli cell sperm cell sperm viability success tissue preparation tissue processing

项目摘要

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.

项目概要从失败的显微外科睾丸活检中分离活的人类精子不孕不育很常见；它影响了 15% 的总人口，有 730 万对美国夫妇每年接受不孕症评估。女性和男性都可能不育。最严重的男性不育的情况是精液中找不到精子（10-20%的情况）；一个条件称为无精症。精子是在睾丸中产生的。据信，大多数男性体内没有精子射精时睾丸中可能仍产生极少量的精子。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。在这个建议，我们将测试此应用程序，以确定是否可以在显微外科手术中找到可用的精子目前可用的治疗方法，已失败。我们首先比较睾丸的酶促处理对睾丸活检进行机械“切碎”的组织，以测试这些组织准备中的哪一个方法将导致精子与体细胞和生殖细胞更好的分离，从而产生更高的产量可用精子的数量。然后，我们将测试是否从睾丸活检中取出体细胞，然后再进行对样本进行分类，将增加从患有严重不育症的男性中取出的精子数量。随后，我们还将使用选定的荧光染料组合测试分选的精子是否对精子活力、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，我们会比较受精潜力、胚胎发育、和在动物模型中使用分选和未分选精子的怀孕率，以确保荧光使用的染料不会对生殖产生任何有害影响。这些拟议的实验将为启动人体临床试验提供必要的信息，我们可以分离活精子进行体外受精，并利用男性的下游分析使用当前可用的治疗方法找不到精子。