Detecting biochemical changes in the pregnant mouse cervix by Raman spectroscopy

描述（由申请人提供）：我们对妊娠和分娩期间宫颈重塑的了解尚不完整，部分原因是缺乏对妊娠期间宫颈发生的生化变化的体内研究。阐明子宫颈成熟的机制可以用来预测早产的发生。直到最近，体内研究方法的侵入性太强，不能作为发现工具，特别是在早产妇女中。该方案将使用体内拉曼光谱，这是一种对胶原蛋白含量、胶原蛋白结构、水合作用、脂质、蛋白质和其他生物分子敏感的光学技术，以非侵入性地研究妊娠期间子宫颈的生物化学。利用光纤体内拉曼光谱，我们最近发现小鼠怀孕期间至少四个重要的拉曼光谱峰存在显著差异，包括脂质、胶原蛋白、酰胺键和富集氨基酸（脯氨酸、酪氨酸）的离散特征。对这些光谱的计算分析产生了对妊娠阶段分类准确率为94%的预测算法。在妊娠结束的2小时窗口内进行的研究确定了预测分娩时间的光谱。这种方法提供了详细的宫颈成熟的实时生物分子图谱，这是目前其他方法无法获得的。在本研究中，我们假设子宫颈早熟的不同机制具有独特的拉曼光谱特征，这些特征对应于早产前潜在的生化和机械变化，这些变化可以在体内检测到。提出了两个具体目的：1)确定正常和异常妊娠和分娩小鼠子宫颈的光谱变化；2)通过比较正常和异常分娩时离体子宫颈拉曼光谱的力学和生化变化，确定宫颈重构的特异性介质。拉曼光谱主要用于疾病的检测。我们的生殖生物学和生物工程团队的合作将利用我们在宫颈组织拉曼分析方面的专业知识来研究怀孕期间宫颈成分的动态变化。宫颈生化的关键因素将被确定。体内拉曼光谱将与生物力学研究和成像质谱相结合，这是一种原位蛋白质组学分析的强大工具，用于检查过早或延迟颈椎重塑的小鼠。总之，这些高度创新的方法将产生深入的宫颈生物学概况，将转化为新的非侵入性方法来检测妇女即将发生的早产。

DESCRIPTION (provided by applicant): Our understanding of cervical remodeling during pregnancy and labor is incomplete, partly due to the lack of in vivo studies on the biochemical changes that occur in the cervix over the course of pregnancy. Elucidation of the mechanisms for cervical ripening could be used to predict the onset of preterm labor. Until recently, in vivo research methods were too invasive to be used as discovery tools, particularly in women who present with preterm labor. This proposal will use in vivo Raman spectroscopy, an optical technique that is sensitive to collagen content, collagen structure, hydration, lipids, proteins, ad other biomolecules to non-invasively investigate the biochemistry of the cervix throughout pregnancy. Using fiber optic in vivo Raman spectroscopy, we recently found significant differences in Raman spectra in at least four important peaks during the course of pregnancy in mice, including discrete signatures for lipids, collagen, amide bonds, and enriched amino acids (proline, tyrosine). Computational analysis of these spectra yielded predictive algorithms with 94% classification accuracy for stage of pregnancy. Studies performed in 2-hour windows at the end of pregnancy identified spectra predictive for the timing of parturition. This approach provides a detailed real-time biomolecular map of cervical ripening that is currently unavailable by other means. In this proposal, we hypothesize that the different mechanisms of premature cervical ripening have unique Raman spectral signatures that correspond to underlying biochemical and mechanical changes that precede preterm birth, which can be detected in vivo. Two Specific Aims are proposed: 1) Determine spectral changes in the cervix of mice with normal and abnormal pregnancy and parturition; 2) Identify specific mediators of cervical remodeling by comparing Raman spectra to mechanical and biochemical changes in the ex vivo cervix during normal and abnormal parturition. Raman spectroscopy has primarily been used for detection of disease. Collaboration between our reproductive biology and bioengineering groups will capitalize on our expertise in Raman analysis of cervical tissues to study dynamic changes in cervix composition during pregnancy. Key elements in cervical biochemistry will be identified. In vivo Raman spectroscopy will be combined with biomechanical studies and imaging mass spectrometry, a powerful tool for in situ proteomic analysis, to examine mice with premature or delayed cervical remodeling. Together, these highly innovative approaches will generate in-depth profiles of cervical biology that will translate into novel non-invasive methods to detect impending premature birth in women.