Detecting biochemical changes in the pregnant mouse cervix by Raman spectroscopy

描述(由申请人提供)：我们对怀孕和分娩期间宫颈重塑的了解是不完整的，部分原因是缺乏对怀孕过程中宫颈生化变化的活体研究。宫颈成熟机制的阐明可用于预测早产的发生。直到最近，体内研究方法还太具侵入性，不能用作发现工具，特别是在有早产的女性身上。这项建议将使用体内拉曼光谱，这是一种光学技术，对胶原含量、胶原结构、水合作用、脂肪、蛋白质和其他生物分子敏感，以非侵入性方式研究怀孕期间宫颈的生物化学。使用光纤活体拉曼光谱，我们最近发现在小鼠怀孕期间至少四个重要峰的拉曼光谱有显著差异，包括脂类、胶原、酰胺键和富含氨基酸(脯氨酸、酪氨酸)的离散特征。对这些频谱的计算分析产生了预测算法，对怀孕阶段的分类准确率为94%。在怀孕结束后的两个小时窗口中进行的研究发现，频谱可以预测分娩的时间。这种方法提供了宫颈成熟的详细实时生物分子图谱，这是目前其他方法无法获得的。在这项建议中，我们假设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。提出了两个特定的目标：1)确定正常和异常妊娠和分娩小鼠宫颈的光谱变化；2)通过将拉曼光谱与正常和异常分娩期间体外宫颈的机械和生化变化进行比较，确定宫颈重塑的特定介质。拉曼光谱主要用于检测疾病。我们的生殖生物学和生物工程团队之间的合作将利用我们在宫颈组织拉曼分析方面的专业知识，研究怀孕期间宫颈成分的动态变化。将确定宫颈生物化学中的关键元素。体内拉曼光谱将与生物力学研究和成像质谱学相结合，成像质谱学是原位蛋白质组分析的强大工具，用于检查过早或延迟宫颈重塑的小鼠。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，以检测即将到来的妇女早产。

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.