Validation of Novel Infertility Biomarker

描述（由申请人提供）：本R21探索性提案解决了美国生殖医学的主要空白，即缺乏可靠、客观的方法来准确诊断人类男性不育症。我们的目标是验证精子特异性硫氧还蛋白SPTRX3作为人类精子质量的新型生物标志物。我们将研究男性不育症患者的精子SPTRX3水平与不孕、自然流产和接受辅助生殖疗法（ART）治疗的女性伴侣意外多胎之间的关系。目前的人类精液分析和诊断方法严重依赖于主观的光显微镜评价。这是不准确的，因为有生育能力和不育男性的精液参数有明显的重叠。客观、自动化的精子分析方法已经存在，但价格昂贵，而且仅限于专门的参考实验室，而不是廉价的医生办公室生育测试。我们已经确定SPTRX3蛋白仅由有缺陷的、形态异常的人类精子携带。我们的初步数据表明，携带sptrx3精子的精液含量增加与抗逆转录病毒治疗后妊娠率降低相一致。相比之下，精液中SPTRX3水平较低的男性进行抗逆转录病毒治疗的夫妇产生的多胎明显更多。这个为期两年的项目有一个明确的目标，即证明精液SPTRX3水平与不孕不育门诊人群中受精失败、自然流产和多胎妊娠的发生之间的统计关系。我们的假设是，SPTRX3水平较低的男性的女性伴侣更有可能通过ART怀孕，更不容易自然流产，但她们也更容易多胞胎。通过自动流式细胞分析和免疫标记精子的荧光显微分析，以及半定量免疫印迹/密度测定技术，将对来自不育夫妇的原始和梯度/游泳纯化精液样品中的SPTRX3含量进行评估。高精液SPTRX3含量的影响将根据治疗结果的基本指标进行评估，包括受精和第一次胚胎卵裂率、成功辅助受精和胚胎移植后自然流产的发生率，以及接受抗逆转录病毒治疗的夫妇多胎妊娠的发生率。为了从机制上理解SPTRX3的携带如何影响人类精子的功能，我们将精子染色质结构（可能受到含有SPTRX3的核空泡的影响）与流式细胞术SPTRX3数据联系起来。我们还将评估高/低SPTRX3样本的精子与仓鼠卵子异源ICSI后的雄性原核发育情况，这是一种通常用于预测人类精子受精能力和发育潜力的方法。根据我们的临床合作者的反馈，其中一位将参与该项目，我们预计基于SPTRX3的测试将使临床医生能够在一般不孕症临床人群中做出宫内人工授精（IUI）与体外受精（IVF/ICSI）之间的治疗决策。在接受体外受精/ICSI治疗的夫妇中，决定应该移植多少胚胎以获得单胎妊娠，而不是不怀孕或不想要的多胎妊娠。在被提议的研究验证后，这种新型生物标志物可以以探针的形式快速转移到临床实践中，用于参考实验室测试，或用于医生办公室使用的横向流动盒（量尺）；还可以开发一种非处方家庭生育测试。

DESCRIPTION (provided by applicant): This R21 exploratory proposal addresses a major gap in U.S. reproductive medicine, namely the lack of reliable, objective methodology for accurate diagnostics of human male infertility. Our goal is to validate the sperm-specific thioredoxin SPTRX3 as a novel biomarker of human sperm quality. We will examine how sperm SPTRX3 levels in male infertility patients correlate to a failure to conceive, to spontaneous abortion and to unwanted multiple births in their female partners treated by assisted reproductive therapies (ART). Current methods of human semen analysis and diagnostics of human male infertility rely heavily on subjective light microscopic evaluation. This is inaccurate because of a significant overlap between semen parameters of fertile and infertile men. Objective, automated methods for sperm analysis exist but are expensive and confined to specialized reference laboratories, as opposed to an inexpensive doctor's office fertility test. We have already established that SPTRX3 protein is carried exclusively by defective, morphologically deviant human spermatozoa. Our preliminary data demonstrate that the increased semen content of SPTRX3-carrying spermatozoa coincides with reduced pregnancy rates after ART. In contrast, ART couples with male partners exhibiting low semen SPTRX3 levels produced significantly more multiple births. This two year project has ONE SPECIFIC AIM, i.e. to demonstrate a statistical relationship between semen SPTRX3 levels and the occurrence of fertilization failure, spontaneous pregnancy loss and multiple pregnancies in couples from a general infertility clinic population. Our HYPOTHESIS is that the female partners of men with low SPTRX3 levels are more likely to conceive by ART and less likely to suffer a spontaneous abortion, but they are also more prone to multiple births. Semen SPTRX3 content will be evaluated in raw and gradient/swim-up purified semen samples from infertile couples by automated flow cytometric analysis and epifluorescence-light microscopic analysis of immunolabeled spermatozoa, and by semi-quantitative western blotting/densitometry technique. The influence of high semen SPTRX3 content will be evaluated against basic measures of treatment outcome, including fertilization and first embryo cleavage rates, incidence of spontaneous abortion following a successful assisted fertilization and embryo transfer, and the incidence of multiple pregnancies in couples treated by ART. To gain mechanistic understanding of how SPTRX3 carryover affects human sperm function, we will correlate sperm chromatin structure, likely affected by the presence of SPTRX3-containing nuclear vacuoles, with flow cytometric SPTRX3 data. We will also assess male pronuclear development after heterologous ICSI of sperm from high/low SPTRX3 samples into hamster ova, an assay commonly used to predict human sperm fertilizing ability and developmental potential. Based on feedback from our clinical collaborators, one of whom will participate in this project, we anticipate that the SPTRX3 based test will allow clinicians to make a treatment decision between intrauterine insemination (IUI) versus IVF/ICSI in the general infertility clinic-population. In the IVF/ICSI treated couples, a decision will be facilitated for how many embryos should be transferred to obtain a singleton pregnancy, as opposed to no pregnancy or an unwanted multiple birth. Upon validation by the proposed research, this novel biomarker can be quickly transferred to clinical practice in the form of probe for a reference laboratory test, or a lateral flow cassette (dipstick) for doctor's office use; an over the counter home fertility test could also be developed. PUBLIC HEALTH RELEVANCE: This project will explore and validate a novel biomarker of human male infertility, the recently discovered sperm borne protein SPTRX3. Preliminary data suggest that this biomarker can be used to more accurately diagnose male infertility and also to predict the likelihood of multiple births and spontaneous pregnancy losses after assisted fertilization. The proposed exploratory research is necessary for future diagnostic technology development leading to the introduction of this innovative diagnostic tool in US infertility clinics. Annually, more than 135,000 infertile couples are treated for infertility in US clinics, where the treatment-decision making still relies on outdated, highly subjective light microscopic method of semen evaluation.