A chemical derivatization-based pseudotargeted liquid chromatography-tandem mass spectrometry method for sensitive and high coverage determination of bile acids in human serum.

Global profiling of bile acids (BAs) is imperative for understand their function and disease pathogenesis. But it is still a challenging task, as the collision-induced dissociation (CID) fragment ions of unconjugated BAs showed low ion intensities to insufficient analysis. Herein, we developed a highly sensitive method for pseudotargeted profiling of BAs by chemical derivatization. In the developed method, a labeling reagent, 2-dimethylaminoethylamine (DMED), was adopted to label the carboxyl group of BAs. The results demonstrated that the detection sensitivities of unconjugated BAs were increased by 4-200 folds after DMED-labeling. Moreover, to profile other potential BAs not included in the 91 known targets, diverse survey experiments were performed on Qtrap-MS to search BAs for both precursor and fragment ion species, and retention index (RI) strategy was adopted to facilitate the identification of isomers. Finally, MRM-based LC-MS/MS method was validated for the pseudotargeted profiling of the BAs submetabolome with good linearity (r2 ≥ 0.990 for 89 known BAs) and high sensitivity (0.05-0.5 ng/mL for unconjugated BAs), covering unconjugated, glycine, taurine, sulfuric acid, glucuronic acid, and as well as those doubly-conjugated with above types. With this method, a total of 107 BAs, covering 54 BAs identified by authentic standards and 53 BAs candidates, were successfully determined in human serum of women with intrahepatic cholestasis of pregnancy (ICP). Multivariate analysis revealed deferentially expressed BAs. ICP disease altered the BAs profile with a reduced proportion of unconjugated, sulfate- and doubly-conjugated BAs and an increased proportion of glycine and taurine conjugates. Altered proportion and profile of BAs in ICP groups were gradually recovered during the ursodeoxycholic acid (UDCA) therapy. Overall, the strategy of DMED-labeling technique combined with diverse survey experiments is sufficiently sensitive and robust to comprehensively analysis of metabolic profiling of BAs in human serum.

Risk Prediction Model of Early-Onset Preeclampsia Based on Risk Factors and Routine Laboratory Indicators.

Background: Globally, 10-15% of maternal deaths are statistically attributable to preeclampsia. Compared with late-onset PE, the severity of early-onset PE remains more harmful with higher morbidity and mortality. Objective: To establish an early-onset preeclampsia prediction model by clinical characteristics, risk factors and routine laboratory indicators were investigated from pregnant women at 6 to 10 gestational weeks. Methods: The clinical characteristics, risk factors, and 38 routine laboratory indicators (6-10 weeks of gestation) including blood lipids, liver and kidney function, coagulation, blood count, and other indicators of 91 early-onset preeclampsia patients and 709 normal controls without early-onset preeclampsia from January 2010 to May 2021 in Peking University Third Hospital (PUTH) were retrospectively analyzed. A logistic regression, decision tree model, and support vector machine (SVM) model were applied for establishing prediction models, respectively. ROC curves were drawn; area under curve (AUCROC), sensitivity, and specificity were calculated and compared. Results: There were statistically significant differences in the rates of diabetes, antiphospholipid syndrome (APS), kidney disease, obstructive sleep apnea (OSAHS), primipara, history of preeclampsia, and assisted reproductive technology (ART) (p < 0.05). Among the 38 routine laboratory indicators, there were no significant differences in the levels of PLT/LYM, NEU/LYM, TT, D-Dimer, FDP, TBA, ALP, TP, ALB, GLB, UREA, Cr, P, Cystatin C, HDL-C, Apo-A1, and Lp(a) between the two groups (p > 0.05). The levels of the rest indicators were all statistically different between the two groups (p < 0.05). If only 12 risk factors of PE were analyzed with the logistic regression, decision tree model, and support vector machine (SVM), and the AUCROC were 0.78, 0.74, and 0.66, respectively, while 12 risk factors of PE and 38 routine laboratory indicators were analyzed with the logistic regression, decision tree model, and support vector machine (SVM), and the AUCROC were 0.86, 0.77, and 0.93, respectively. Conclusions: The efficacy of clinical risk factors alone in predicting early-onset preeclampsia is not high while the efficacy increased significantly when PE risk factors combined with routine laboratory indicators. The SVM model was better than logistic regression model and decision tree model in early prediction of early-onset preeclampsia incidence.

Predictive value of the complement factors B and H for women with gestational diabetes mellitus who are at risk of preeclampsia.

OBJECTIVES: We aimed to define the relationships between the serum concentrations of complement factors B and H in mid-pregnancy and the risk of preeclampsia (PE) in patients with gestational diabetes mellitus (GDM). STUDY DESIGN: We performed a prospective nested case-control study of 503 patients with GDM who attended Peking University Third Hospital between March 2018 and December 2018. 456 patients were followed until delivery and blood samples were collected between gestational weeks 24 and 28. Thirty patients developed PE, 12 developed gestational hypertension (GH), and 42 matched cases were selected as a control group. RESULTS: The incidence of PE was 5.96 %. The serum concentrations of triacylglycerol (TG), FB, and FH of women with GDM who developed PE (GDM-PE group) in mid-pregnancy were significantly higher than those of the GDM group [TG: 3.60 (2.94-4.63) vs 2.54 (2.14-3.01) mmol/L, p < 0.001; FB: 346 (314-378) vs 284 (263-323) mg/L, p < 0.001; FH: 417 ± 45 vs 379 ± 47 mg/L, p = 0.003]. Multivariate regression analysis showed that high serum concentrations of TG and FB in mid-pregnancy were related to the risk of patients with GDM developing PE [TG: odds ratio (OR) 2.035 (95 % confidence interval (CI) 1.032-4.013); FB: OR 1.018 (95 % CI 1.001-1.035)]. The area under the curve (AUC) of FB for the prediction of PE was 0.821 (95 % CI 0.722-0.921) and that for a combination of TG, FB, and FH was 0.857 (95 % CI 0.770-0.944). CONCLUSIONS: High serum FB concentration during mid-pregnancy is a potential predictor of the risk of PE in patients with GDM, and the combination of FB, FH, and TG increased this predictive value.

Expression of Matrix Metalloproteinase-2,-7,-9 in Serum during Pregnancy in Patients with Pre-Eclampsia: A Prospective Study.

BACKGROUND: Matrix Metalloproteinases (MMPs) have been found to have important roles in vascular pathology and may be involved in the occurrence of pre-eclampsia. In this study, the serum levels of MMP-2, -7, -9 in normal pregnant women and pre-eclampsia patients were analyzed to assess their predictive value. METHODS: A total of 1563 pregnant women from Peking University Third Hospital, from February 2021 to October 2021, were enrolled. Serum samples were collected from patients one to three times, during the different trimesters. Among the 102 singleton pre-eclampsia patients, we collected samples from 33 patients in the first trimester (6-13 GW), 33 in the second trimester (14-28 GW), 41 in the third trimester (29-41 GW) and 28 after onset of pre-eclampsia. Samples from each trimester were collected before the onset of pre-eclampsia. Then we selected 35, 37, 43 and 25 samples from 124 healthy pregnant women by matching their age, BMI and gestational weeks, using these as the control groups. Serum levels of MMP-2, -7, -9 were detected by ELISA. The receiver operating characteristic (ROC) curve was used to evaluate their predictive value. RESULTS: Except for the first trimester, MMP-2 and MMP-7 were significantly higher in the pre-eclampsia group (p < 0.5). Additionally, in the pre-eclampsia group, MMP-9 increased significantly in the first trimester and after the onset of pre-eclampsia but decreased significantly in the second and third trimesters (p < 0.5). The ROC curve indicated that MMP-9, MMP-2 and MMP-7 were the best indicators for predicting pre-eclampsia in the first, second and third trimesters, respectively. CONCLUSION: Increased MMP-2 and MMP-7 levels and a decreased MMP-9 level seem to be related to the pathogenesis of pre-eclampsia and are expected to be potential predictors of pre-eclampsia.

The correlation between maternal serum sST2, IL-33 and NT-proBNP concentrations and occurrence of pre-eclampsia in twin pregnancies: A longitudinal study.

The primary objective of this study was to determine the longitudinal profile of serum sST2 (soluble suppression of tumorigenicity 2), IL-33 (interleukin-33) and NT-proBNP (N-terminal pro-brain natriuretic peptide) concentrations in twin pregnancies with pre-eclampsia (PE) and those normotensive twins. The secondary objective was to test whether the change of serum sST2,IL-33 and NT-proBNP is related to PE in twin pregnancies. This is a longitudinal nested case-control study and all 156 dichorionic (DC) pregnancies were from a prospective cohort of twin pregnancies who received antenatal care and gave two live births at Peking University Third Hospital between October 2017 and September 2020. Four to five milliliters of peripheral blood of each pregnant woman were collected during the following three intervals: (1) 6-11+6  weeks; (2) 24-27+6  weeks; (3) 28-31+6  weeks. We found that sST2 and NT-proBNP levels increased as pregnancy progressed in normotensive twin pregnancies and further increased in PE group, while no differences were found in IL-33 levels throughout pregnancy. Then the correlation of biomarker levels with the occurrence of PE was assessed. Our results indicated that combining maternal serum sST2 and NT-proBNP levels yielded the highest predictive value on the occurrence of PE significantly higher than the predictive value of any markers alone. Interestingly, the predictive value of second trimester (AUC = 0.876, 95%CI 0.824-0.928, LR-0.338, LR+7.67, p < 0.001)was higher than that of early-third trimester (AUC = 0.832, 95%CI 0.769-0.896, LR-0.29, LR+3.845, p < 0.001). Serum sST2 and NT-proBNP concentrations during second and early-third trimester were associated with the occurrence of PE in twin pregnancies.

Correlation of low fetal fraction of cell-free DNA at the early second-trimester and pregnancy complications related to placental dysfunction in twin pregnancy.

Introduction: This study aimed to determine the correlation between fetal fraction (FF) of cell-free DNA (cf-DNA) and pregnancy complications related to placental dysfunction in Twin Pregnancy. Methods: This retrospective cohort study analyzed twin pregnant women who underwent non-invasive prenatal testing (NIPT) at 12+0-26+6 weeks of gestation from April 2017 to April 2021. Low fetal fraction (LFF) was defined individually as less than the 25th, 10th, 5th, and 2.5th percentile among all fetal fractions in the cohort. Primary outcomes included gestational hypertension (GH), preeclampsia (PE), gestational diabetes mellitus (GDM), and small for gestational age (SGA). Logistic regression analysis was used to assess the relationship between LFF and pregnancy complications. Results: A total of 500 twin pregnancies (male-male twins, 245; female-female twins, 255) were included in this study. In LFF group (FF < 25th percentiles), maternal BMI was significantly higher than FF > 75th percentiles (23.6 kg/m2 vs. 21.3 kg/m2; P < 0.001). The risk of SGA increased gradually from FF < 25th percentiles [adjusted odds ratio (OR), 1.71; 95% confidence interval (CI), 1.07-2.99; P = 0.016] to FF < 2.5th percentiles (adjusted OR, 4.44; 95% CI,1.33-14.82; P < 0.015). In addition, the risks of SGA in both fetuses were higher than the risks of at least one fetus SGA in LFF group. LFF had no correlation with GH, PE, and GDM in twin pregnancy. Conclusion: LFF has a strong association with increased risk of SGA in twin pregnancy. Moreover, FF of cf-DNA may provide a new idea for the early screening of diseases related to placental dysfunction in twin pregnancy.

Maternal lipid levels in preeclampsia: singleton vs. twin pregnancies.

OBJECTIVE: To compare the maternal lipid levels in preeclampsia (PE) patients between singleton and twin pregnancies. METHODS: In this retrospective study, pregnant women with PE were divided into singleton group (n = 702) and twin group (n = 198). Serum lipids which include total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured and the TC/HDL-C ratio, TG/HDL-C ratio, and LDL-C/HDL-C ratio were calculated and compared between the two groups. Covariance analysis was used to correct the potential factors affecting serum lipid levels such as maternal age, pre-pregnancy body mass index, gestational weight gain, etc. RESULTS: The levels of TC, TG, LDL-C, and TC/HDL-C ratio, TG/HDL-C ratio, LDL-C/HDL-C ratio in twin PE were significantly higher than those in singleton PE group, and there was no significant difference in the level of HDL-C between the two groups. In late-onset PE patients, the lipid levels of TC, TG, LDL-C, and TC/HDL-C ratio, TG/HDL-C ratio, LDL-C/HDL-C ratio in twin PE group were significantly higher than those in singleton PE group, with no significant difference in the level of HDL-C. However, in early-onset PE patients, there were no significant differences in the lipid levels between the two groups. CONCLUSIONS: There were more obvious lipid disturbances such as higher levels of TC, TG, LDL-C, and TC/HDL-C ratio, TG/HDL-C ratio, LDL-C/HDL-C ratio in twin PE group than singleton PE group. The differences of lipid levels appeared mainly in late-onset PE group, while the lipid levels in twin PE group were similar to those in singleton PE group during pregnancy in early-onset PE group.

Gut Microbiota Dysbiosis and Increased Plasma LPS and TMAO Levels in Patients With Preeclampsia.

Objective: To characterize the gut microbiota in patients with preeclampsia (PE) compared with healthy controls. Methods: We analyzed and compared the microbiota communities in the feces of 48 PE patients with 48 age-, gestational weeks-, and pre-pregnancy body mass index-matched healthy controls using 16S rRNA gene sequencing, and also we tested fecal and plasma lipopolysaccharide (LPS) and plasma trimethylamine-N-oxide (TMAO) concentration levels in the two groups. Results: Compared with the control group, microbial alpha diversity was lower in the PE group, but there was no statistically significant difference between the two groups. At the phylum level, Firmicutes (51.64% PE vs. 59.62% Control, P < 0.05), Bacteroidetes (40.51% PE vs. 34.81% Control, P< 0.05), Proteobacteria (4.51% PE vs. 2.56% Control, P < 0.05), and Actinobacteria (2.90% PE vs. 1.77% Control, P < 0.05), exhibited significant differences between the PE group and the control group. LEfSe analysis found 17 differentially abundant taxa between the two groups. PICRUSt analysis found that in the KEGG pathways, the microbial gene functions related to LPS biosynthesis were higher in the fecal microbiome of the PE group. The fecal and plasma LPS concentrations and plasma TMAO concentrations of PE patients were higher than those of the healthy controls. Conclusion: PE patients had gut microbiota dysbiosis and increased plasma LPS and TMAO levels, which will lead to a better understanding of the relationship between the gut microbiota and PE.

Efficacy of Different Treatment Regimens for Antiphospholipid Syndrome-related Recurrent Spontaneous Abortion.

BACKGROUND: Antiphospholipid syndrome (APS)-related immune factors are considered as an important cause of recurrent spontaneous abortion (RSA). Anticoagulant and anti-inflammatory treatments are believed to effectively improve adverse pregnancy outcomes by affecting the abnormal autoimmune response of the maternal-fetal interface. The aim of this study was to observe the clinical characteristics and treatment outcomes of anticoagulant regimens and anti-inflammatory plus anticoagulation regimens for APS-related RSA. METHODS: APS-related RSA cases from September 2011 to September 2016 at Peking University Third Hospital were retrospectively analyzed. The patients were assigned to study group (anti-inflammation plus anticoagulation) and control group (simple anticoagulation). The incidence of repeat abortion, the incidence of placental dysfunction, the gestational weeks of pregnancy, and the mean weight of the fetus were observed. RESULTS: The pregnancy and neonatal outcome indicators of the repeat pregnancy loss rate (11.11% vs. 22.70%), placental dysfunction-related diseases (6.35% vs. 15.60%), the mean birth weight of infants born after 24 weeks gestation (3152.41 ± 844.67 g vs. 2765.76 ± 816.40 g), full-term delivery weight (3456.28 ± 419.79 g vs. 3076.18 ± 518.79 g), the proportions of low birth weight infants (12.70% vs. 21.98%), and small for gestational age (6.35% vs. 14.18%) differed significantly between the study and control groups (all P< 0.05). The incidence of preterm delivery, term delivery, and stillbirth was not significantly different between the two groups, and there was no significant difference between the study and control groups in gestational age at birth (37.6 ± 3.3 weeks vs. 36.9 ± 3.2 weeks; P > 0.05). CONCLUSION: The anti-inflammatory and anticoagulation regimen is more effective than the simple anticoagulation regimen in the treatment of APS recurrent abortion.

Co-morbidity of cervical incompetence with polycystic ovarian syndrome (PCOS) negatively impacts prognosis: A retrospective analysis of 178 patients.

BACKGROUND: Cervical incompetence is an important cause of miscarriage and premature birth and polycystic ovary syndrome is a heterogeneous endocrine disorder that is the most common cause of anovulatory infertility and eugonadotrophic hypogonadism. By now, it is still debated whether women with PCOS have an increased risk of miscarriage and there have been no studies about the pregnancy outcomes of cervical incompetence patients with PCOS. METHODS: The following clinical data of cervical incompetence patients with/without PCOS who were treated between September 2006 and September 2013 were retrospectively analysed: onset gestational age, termination gestational age, pregnancy outcome, co-morbid insulin resistance (IR) in PCOS patients, the influence of IR, co-morbid hyperandrogenism (HA) in PCOS patients, and the influence of HA. The independent samples t-test and chi-square trend test were used to analyse the data. RESULTS: A total of 178 singleton pregnancy cases with cervical incompetence were identified. The average onset gestational age was 23.9 ± 4.3 weeks, and the average termination gestational age was 32.5 ± 5.5 weeks. Of these 178 singleton pregnancy cases, 40 (22.5 %) ended in miscarriage, 82 (46.1 %) ended in preterm birth, and 56 (31.5 %) ended in term birth. Eighty cases (44.9 %) exhibited PCOS co-morbidity, and those cases had an average onset gestational age of 22.3 ± 3.8 weeks and an average termination gestational age of 31.2 ± 5.7 weeks, which were both significantly different from those of the non-PCOS group (both P < 0.001). Compared with the non-PCOS group (15.3 % miscarriage, 48.0 % preterm birth, and 36.7 % term birth), the PCOS group exhibited worse pregnancy outcomes (31.3 % miscarriage, 43.8 % preterm birth, and 25 % term birth) (P = 0.01). Among the 80 PCOS patients, 45 (56.3 %) exhibited co-morbid IR, and the IR group exhibited significantly worse pregnancy outcomes than the non-IR group (P = 0.03). Among the 80 PCOS patients, 54 cases (67.5 %) exhibited co-morbid HA, and there was no statistical difference on the pregnancy outcomes between the two groups. The multivariate logistic regression model revealed that PCOS was significantly correlated with miscarriage (OR: 3.72, 95 % CI: 1.37-10.13). CONCLUSIONS: The cervical incompetence patients with co-morbid PCOS exhibited earlier onset gestational ages, earlier termination gestational ages and worse pregnancy outcomes. For patients with co-morbid insulin resistance, the pregnancy outcomes were worse than expected.

[Serum relaxin in cervical incompetence patients].

OBJECTIVE: To analyze the serum relaxin and clinical character of cervical incompetence patients and normal pregnant women. METHODS: A total of 33 cervical incompetence patients (research group) and 33 normal pregnancy women with the same gestational age (control group) were recruited into the study. The serum relaxin level was detected with enzyme labeled immunosorbent assay (ELSIA) in the two groups, and the cervical length of early pregnancy period (cm), body mass index (BMI, kg/m2), frequency of polycystic ovary syndrome (%), gestational diabetes mellitus/diabetes mellitus (%) and outcomes in the two groups were analyzed with independent samples t test and chi-square test. RESULTS: All the cervical incompetence patients were recruited between Feb. 2008 and Sept. 2012, with the average termination gestational age of 30±6 weeks. Among them, 15 (45.45%) was abortion, 12 (36.36%) was preterm birth, 6 (16.18%) was term birth. The average BMI before pregnancy was 27±4 kg/m2, and the average serum relaxin was 2,748±82 mg/L; for the 33 patients in the control group, the average termination gestational age was 38±3 weeks, and 1 (3.03%) of them was abortion, 4 (12.12%) was preterm birth, 28 (84.85%) was term birth. The average BMI before pregnancy was 23±3 kg/m2, the average serum relaxin was 2,602±126 mg/L. Compared with the control group, the research group had more patients who complicated with polycystic ovary syndrome and gestational diabetes mellitus/diabetes mellitus (P<0.01, <0.05) and worse pregnancy outcomes (P<0.01); the average BMI before pregnancy and the average serum relaxin level of the research group were significantly higher than control group (P<0.01, P<0.01). Analysis through the unconditional logistic regression showed that BMI and serum relaxin were both independent risk factors of cervical incompetence. CONCLUSIONS: The high level of serum relaxin is an independent risk factor of cervical incompetence; women with polycystic ovary syndrome may more likely to have cervical incompetence and serum relaxin may have the predictive value for cervical incompetence.