Assay development for the assessment of pregnancy risks in early pregnancy

关于早期人类胎盘形成的临床信息缺乏，因为许多妊娠病理 起源。为了解决这一差距，并开始开发强大的诊断工具来管理怀孕 并发症：我们建议进行一项初步研究，以分析胎盘滋养层细胞中的疾病特异性蛋白。vbl.使用 一种安全宫颈涂片在怀孕5到20周之间我们可以非侵入性地捕获数百种同质的， 表达HL A-G-和hCG的滋养层细胞。我们认为这些胎盘细胞对评估 体内妊娠状况和围产期疾病风险评估。我们的前提是基于我们公布的数据 证明获得的分离的胎盘细胞表达额外的绒毛滋养细胞谱系标记(例如， 人绒毛膜促性腺激素、人类白细胞抗原G)，并具有与病理相关的分子图谱。免疫细胞化学(ICC)蛋白不包括 对细胞的压力分析表明，怀孕期间几种关键蛋白质的水平发生了变化 出现流产、胎儿生长受限(FGR)或先兆子痫。由于所应用的方法很难 转化为临床工具(标准化和多路复用功能)，我们现在将使用强大和高度敏感的- 不需要事先从临床样本中分离细胞的阳性单细胞质量细胞术分析。我们 假设，根据我们发表的研究中获得的可靠的ICC数据，AFP和PGF水平是 妊娠EVT细胞显著改变与FGR有关，FGR是以胎盘为基础的围产儿的关键指标 精神错乱。因此，我们将严格确定在我们的初步研究中发现的这些蛋白质的表达 对宫颈标本中胎儿细胞的研究与胎儿生长速度相关，考虑到胎儿性别和其他 相关因素。这项应用的具体目的是建立颈椎的基本关系 绒毛外滋养细胞生物标志物水平对妊娠结局的影响。我们将量化AFP和PGF，并 比较妊娠早期宫颈标本中胎盘细胞的细胞类型特异性蛋白 正常足月妊娠的受试者与低出生体重GA相关的不良结局的受试者 在出生的时候。这一目标将在三个里程碑中实现。里程碑1，我们将验证一个更高的高度- 灵敏的单细胞质谱法测定滋养层细胞中AFP和PGF及其最佳值 将检测结果与临床标本比对。里程碑2，我们将收集多达100个滋养细胞样本 在怀孕的前三个月，连同未确认的医疗记录一起确定妊娠结局。里程碑3， 其余的患者滋养细胞样本将被检测AFP和PGF和其他蛋白质水平和 与患者结局相比，特别是降低了胎龄的出生体重。这些实验将 验证测试平台并为更大规模的临床研究提供初步数据，以确定围产期风险 分数和胎盘紊乱的测试。我们的长期目标是开发检测试剂盒并将其商业化 安全获得的EVT细胞用于围产期病理早期诊断的独特IP产品 持续怀孕。构建此测试将允许创建风险分数，从而能够进行识别 这将有助于减轻患者的心理压力，并有助于转移 医疗保健负担给有需要的患者。

There is a lack of clinical information available on early human placentation when many pregnancy pathologies originate. To address this gap and to begin the development of robust diagnostic tools to manage pregnancy complications we propose a pilot study to analyze disease specific proteins in placental trophoblast cells. Using a safe pap smear between 5 to 20 wks of gestation we can non-invasively capture hundreds of homogeneous, HLA-G-and hCG expressing trophoblast cells. We propose that these placental cells are useful for assessing pregnancy status and assessing risk of perinatal disease in vivo. Our premise is based on our published data demonstrating that isolated placental cells obtained express extra villous trophoblast lineage markers (e.g., hCG, HLA-G), and have molecular profiles associated with pathology. Immunocytochemical (ICC) protein ex- pression analysis of the cells demonstrates altered levels of several key proteins in pregnancies that later develop miscarriage, fetal growth restriction (FGR) or preeclampsia. Since the applied method are difficult to translate into a clinical tool (standardization and multiplex capabilities), we will use now a robust and highly sen- sitive single cell mass cytometry assay that does not require prior cell isolation from the clinical sample. We hypothesize that, based on robust ICC data obtained in our published studies, that AFP and PGF levels are significantly altered in EVT cells from pregnancies linked to FGR, a key indicator of placenta-based perinatal disorders. Therefore, we will rigorously determine if expression of these proteins identified in our preliminary studies in fetal cells from cervical specimens correlate with fetal growth rates, considering fetal sex and other relevant factors. The specific aim of this application is to establish the fundamental relationship of cervical extra-villous trophoblast biomarker levels to pregnancy outcomes. We will quantify AFP and PGF and cell type specific proteins in placental cells in cervical specimens in the first trimester, comparing cohorts of subjects with normal term pregnancies to those with adverse outcomes associated with low birth weight for GA at birth. This goal will be accomplished in three milestones. Milestone 1, we will validate a superior high- sensitivity single cell mass spectrometry assay (MSA) to quantify AFP and PGF in trophoblast cells and opti- mize the assay with clinical specimens. Milestone 2, we will collect up to 100 specimens of trophoblast cells in the first trimester, along with de-identified medical records to determine pregnancy outcomes. Milestone 3, the remaining patient trophoblast samples will be assayed for AFP and PGF and other protein levels and compared to patient outcomes, particularly reduced birth weight for gestational age. These experiments will validate the testing platform and provide preliminary data for a larger clinical study to establish a perinatal risk scores and a test for placental disorders. Our long-term goal is to develop and commercialize testing kits and products with unique IP for early diagnosis of perinatal pathologies using EVT cells obtained safely during ongoing pregnancies. Building this assay will allow the creation of risk scores that will enable the identification of high as well as low risk patients that will aide to reduce psychological stress for patients and help shifting healthcare burden to patients in need.