Delivery prediction by quantitative analysis of four steroid metabolites with liquid chromatography tandem mass spectrometry in asymptomatic pregnant women

Prediction of delivery is important for assessing due dates, providing adequate prenatal care, and suggesting appropriate interventions in preterm and post-term pregnancies. Recent metabolomic findings suggested that the temporal abundance information of metabolome can be used to predict delivery timing with high accuracy in a cohort of healthy women. However, a targeted and quantitative assay is required to further validate the clinical performance and utility of this group of metabolomic candidates in delivery prediction with a larger and independent cohort. LC-MS/MS quantitative assays were applied to determine the plasma concentrations of four steroid metabolites, including oestriol-16-glucuronide (E3-16-Gluc), 17-alpha-hydroxyprogesterone (17-OHP), tetrahydrodeoxycorticosterone (THDOC), and androstane-3,17-diol (A-3,17-Diol) in asymptomatic women of singleton pregnancies (≥30^th gestational weeks). Subsequent statistical analysis was conducted to assess the performance of the above candidates in delivery prediction. Using LC-MS/MS, four steroids were separated and quantified in 5.5 min. The coefficients of variation (CVs) of the four analytes at the lower limit of quantification ranged from 7.9% to 14.6%, with the <i>R</i>² values greater than 0.990 in the calibration curves. Of the 585 recruited pregnant women who ended up with spontaneous delivery, 17.1% and 82.9% of the subjects delivered within and after 7 days since plasma collection, respectively. In the receiver operator curve analysis, the gestational age-adjusted area under the curve of the combined measurements of E3-16-Gluc and 17-OHP was 0.69 (95% CI: 0.60–0.76), with the sensitivity of 87.0% (95% CI: 78.8%–92.9%) and specificity of 60.2% (95% CI: 55.7%–64.6%). Moreover, the positive and the negative predictive values were 28.3%–34.0% and 93.1%–97.4% respectively for this combined panel. We performed analytical and clinical validation of a quantitation LC-MS/MS panel for the four steroids in the plasma of pregnant women. The steroid metabolites panel of E3-16-Gluc and 17-OHP was potentially useful for predicting delivery within one week in asymptomatic women of singleton pregnancies.
Key messagesA quantitative LC-MS/MS assay for determining the plasma levels of 17-OHP, THDOC, A-3,17-Diol and E3-16-Gluc was developed and validated, in order to evaluate their predictive performance in asymptomatic delivery of singleton pregnancy. The levels of E3-16-Gluc and 17-OHP were found to be significantly elevated at the time of sampling in women that delivered within one week and their combinational testing may be potentially useful in delivery prediction. A quantitative LC-MS/MS assay for determining the plasma levels of 17-OHP, THDOC, A-3,17-Diol and E3-16-Gluc was developed and validated, in order to evaluate their predictive performance in asymptomatic delivery of singleton pregnancy. The levels of E3-16-Gluc and 17-OHP were found to be significantly elevated at the time of sampling in women that delivered within one week and their combinational testing may be potentially useful in delivery prediction.

分娩预测对于评估预产期、提供充足产前护理，以及为早产与过期妊娠患者制定合理干预方案均具有重要意义。近期代谢组学研究表明，在健康女性队列中，代谢组的时序丰度信息可用于高精度预测分娩时间。然而，仍需通过靶向定量检测手段，在更大规模的独立队列中进一步验证该类代谢组候选标志物在分娩预测中的临床效能与应用价值。

本研究采用液相色谱-串联质谱法（LC-MS/MS）定量检测孕周≥30周的单胎妊娠无症状女性血浆中4种类固醇代谢物的浓度，包括雌三醇-16-葡萄糖苷酸（oestriol-16-glucuronide, E3-16-Gluc）、17-α-羟孕酮（17-alpha-hydroxyprogesterone, 17-OHP）、四氢脱氧皮质酮（tetrahydrodeoxycorticosterone, THDOC）以及雄烷-3,17-二醇（androstane-3,17-diol, A-3,17-Diol）。后续通过统计学分析评估上述候选标志物在分娩预测中的应用效能。

采用该LC-MS/MS方法可在5.5分钟内完成4种类固醇的分离与定量检测。在定量下限浓度下，4种分析物的变异系数（CVs）介于7.9%至14.6%之间，校准曲线的决定系数（R²）均大于0.990。本研究共招募585例最终自然分娩的孕妇，其中17.1%的受试者在血浆采样后7天内分娩，82.9%的受试者在采样7天后分娩。

受试者工作特征曲线（ROC）分析结果显示，联合检测E3-16-Gluc与17-OHP的指标经孕周校正后的曲线下面积（AUC）为0.69（95%置信区间：0.60–0.76），灵敏度为87.0%（95%置信区间：78.8%–92.9%），特异度为60.2%（95%置信区间：55.7%–64.6%）。此外，该联合检测组合的阳性预测值与阴性预测值分别为28.3%–34.0%与93.1%–97.4%。

本研究完成了针对孕妇血浆中4种类固醇的LC-MS/MS定量检测体系的分析与临床验证。针对E3-16-Gluc与17-OHP的类固醇代谢物联合检测组合，可用于预测单胎妊娠无症状女性1周内的分娩事件，具备潜在应用价值。

【核心要点】
本研究开发并验证了一种可定量检测孕妇血浆中17-OHP、THDOC、A-3,17-Diol及E3-16-Gluc水平的LC-MS/MS方法，以评估其在单胎妊娠无症状分娩预测中的效能。研究发现，在采样后1周内分娩的孕妇中，E3-16-Gluc与17-OHP的血浆水平在采样时显著升高，二者联合检测可用于分娩预测，具备潜在应用价值。本研究开发并验证了一种可定量检测孕妇血浆中17-OHP、THDOC、A-3,17-Diol及E3-16-Gluc水平的LC-MS/MS方法，以评估其在单胎妊娠无症状分娩预测中的效能。研究发现，在采样后1周内分娩的孕妇中，E3-16-Gluc与17-OHP的血浆水平在采样时显著升高，二者联合检测可用于分娩预测，具备潜在应用价值。