Analysis of metabolites in serum and villous tissue of missed abortion patients based on targeted metabolomics

Missed abortion, defined as the cessation of embryo development or death in the uterine cavity during early pregnancy, with the embryo remaining in the uterus without being naturally expelled, is one of the most common adverse pregnancy outcomes. In recent years, the incidence of missed abortion has been on the rise, accounting for about 15% of natural miscarriages in clinical pregnancies ( 1 ).Missed abortion is primarily diagnosed by ultrasonography. Typical ultrasonic features involve irregular gestational sac shape, absent fetal heart activity, and arrested embryonic development. Serial monitoring of serum HCG can also be used for auxiliary diagnosis, with affected patients presenting with slowly increasing or progressively decreasing HCG concentrations. It is generally believed that missed abortion has complex and diverse causes and may result from a combination of multiple factors. Studies have found that fetal chromosomal abnormalities, including numerical and structural aberrations, are the main causative factors for missed abortion. Among these, chromosomal aneuploidy is a significant cause of missed abortions ( 2 ). Moreover, the abnormal expression of maternal autoimmune antibodies such as anti-sperm antibodies and anti-cardiolipin antibodies is often closely related to miscarriage. Research has also found that an abnormal increase in Ureaplasma urealyticum and Chlamydia trachomatis in the female reproductive tract can also be a significant cause of early miscarriages. In addition, factors such as age, bad life habits and other external influences can lead to missed abortion in pregnant women ( 3 ). However, despite being commonly associated with factors such as immune, genetic, infectious, environmental, endocrine, and socio-psychological influences, the cause remains unknown in 50% of cases of missed miscarriages. Therefore, conducting thorough and comprehensive research into the causes of missed abortion and finding more accurate preventive and diagnostic indicators is an urgent task. Metabolomics, which profiles small-molecule metabolites reflecting real-time physiological and pathological states, has emerged as a powerful tool in obstetric research, particularly for gestational diabetes, assisted reproductive technologies, polycystic ovary syndrome, endometriosis, ovarian cancer biomarkers, and preeclampsia ( 4 – 12 ). Among studies related to abortion and metabolism, there are reports indicating differences in plasma metabolites between patients with ectopic pregnancy and those with intrauterine pregnancy abortions. Studies have also shown differences in the metabolites found in the serum and plasma of pregnant women with missed abortions compared to those undergoing normal induced abortions ( 12 – 14 ). However, existing studies are limited to single matrices (serum/plasma) and lack paired serum–villous tissue validation, leaving unclear whether metabolic disturbances are systemic or localized at the maternal–fetal interface. To address these limitations, this study performed targeted metabolomics analysis of paired serum and villous tissue samples from women with missed abortion and normal early pregnancy. We aimed to explore systemic and local metabolic perturbations, identify key differential metabolites, and assess the discriminatory capacity of metabolite combinations. The findings may provide insights into the metabolic dysregulation underlying missed abortion and serve as a reference for further mechanistic studies.

A total of 153 subjects were included in this study, including 78 cases in the missed abortion group and 75 cases in the control group. This study included the general baseline data of patients in the missed abortion group and the control group: age, BMI, height, weight, average diameter of the gestational sac, and days of amenorrhea. Compared with women in the control group, those with the missed abortion group had a higher proportion of women aged < 35 years (98.72% vs. 80.00%), a higher BMI (22.43 kg/m² vs. 21.55 kg/m²), a larger mean gestational sac diameter (24.69 mm vs. 18.49 mm), and a longer duration of amenorrhea (67.45 days vs. 46.09 days) ( Table 1 ). Patient demographics and baseline characteristics. Mean ± SD; n (%). Welch Two Sample t-test. Pearson’s Chi-squared test. Fisher’s exact test. Targeted metabolomics was performed to quantify 11 amino acids and 31 acylcarnitines in the serum and villous tissues from missed abortion and control group subjects. Compared with control group, missed abortion patients showed higher levels of ALA, ARG, CIT, LEU+ILE+ProOH, ORN, PHE, PRO, TYR, VAL, C3, C5 and C18, and lower levels of C4DC/C5OH, C6, C12, C14, C14OH and C16OH (P < 0.05). After false discovery rate (FDR) correction, significant differential expression was observed for 17 metabolites between the missed abortion group and the control group (FDR < 0.05), whereas the other 25 metabolites displayed no statistically significant intergroup differences (FDR ≥ 0.05). Among these differentially expressed metabolites, amino acids including ORN, TYR, LEU+ILE+ProOH, ALA, VAL, PRO, CIT, ARG, and PHE exhibited elevated levels in the missed abortion group, with ORN, TYR, and LEU+ILE+ProOH exhibiting the most pronounced alterations. Additionally, certain carnitines C6, C16OH, C14, and C12 showed reduced levels in the missed abortion group, while other carnitine species, namely C3, C4DC/C5OH, C5, and C14OH, also presented significant intergroup expression differences ( Table 2 ; Figure 1A ). Differential metabolites in the serum. Mean ± SD; n (%). Welch Two Sample t-test. Mann-Whitney U test. (A) Volcano plot for differential metabolites in serum. (B) Volcano plot for differential metabolites in villous tissues.The abscissa represents log 2 (fold change), and the ordinate represents -log 10 (FDR). Red dots indicate metabolites significantly elevated in the missed abortion group (FDR 1), blue dots represent significantly decreased metabolites (FDR < 0.05 and FC < 1), and gray dots denote metabolites with no statistically significant difference. The horizontal dashed line indicates the significance threshold of FDR = 0.05, and the vertical middle line represents the change threshold of log 2 FC = 0. A total of 25 metabolites were identified to be significantly differentially expressed (FDR < 0.05), whereas no statistically significant differences were detected in the expression levels of the other 17 metabolites between the two groups. Most acylcarnitines, such as C16OH, C5DC/C6OH, C10:1, C18, C14OH, C12, C14 and C18OH, were significantly elevated in the missed abortion group, and C10:1 had the highest fold change of 2.448. By contrast, metabolites including C3, C0, C4 and C5 were markedly reduced in the missed abortion group. Regarding amino acid metabolites, ARG and CIT were remarkably increased in the missed abortion group (FDR < 0.05). ORN showed a marginally significant difference (FDR = 0.056) with lower levels compared with the control group. No significant intergroup differences were found in other amino acids, namely ALA, GLY, LEU+ILE+ProOH, MET, PHE, PRO, TYR and VAL ( Table 3 ; Figure 1B ). Differential metabolites in villous tissue. Mean ± SD; n (%). Mann-Whitney U test. Due to complete data separation of carnitine metabolites between the two groups, a stable logistic regression model could not be established; therefore, only the intergroup comparison results were presented. For multivariate regression analysis, only amino acid metabolites with model convergence were included for independent correlation analysis. The adjusted odds ratios (ORs) of the 8 amino acid metabolites were all greater than 1, indicating that elevated serum levels of these metabolites were independently associated with an increased risk of missed abortion, acting as independent risk factors ( Table 4 ). Firth penalized logistic regression analysis of metabolites in serum. Adjusted for age, BMI. Univariate analysis demonstrated that C5DC/C6OH, C6, C3DC/C4OH,CIT and ARG were risk factors for missed abortion (OR>1, P<0.05). While C3, C0,C5, C4 and C4DC/C5OH acted as protective factors (OR<1, P<0.05) ( Table 5 ). Firth penalized logistic regression analysis of metabolites in villous tissue. Adjusted for age, BMI. ROC curve analysis revealed that the top 8 metabolites with the most significant differences after FDR correction exhibited satisfactory discriminatory efficiency for missed abortion, with the area under the curve (AUC) ranging from 0.693 to 0.777. Ornithine (ORN) yielded the highest diagnostic efficiency (AUC = 0.777, 95%CI: 0.704-0.849), followed by hexanoylcarnitine (C6, AUC = 0.763, 95%CI: 0.685-0.840) and tyrosine (TYR, AUC = 0.753, 95%CI: 0.677-0.829). These findings indicated that amino acid and carnitine metabolites possess potential diagnostic values, and can serve as auxiliary biomarkers to distinguish missed abortion from normal early pregnancy. Furthermore, a combined diagnostic model was established based on the eight metabolites. The combined model achieved an AUC of 0.854 (95%CI: 0.797-0.911), which was significantly superior to that of any single metabolite. It suggested that this combined model has favorable auxiliary diagnostic performance and could effectively differentiate missed abortion from normal early pregnancy ( Table 6 ; Figures 2A, C ). AUCs and 95% CI (Serum). (A) ROC curves of the top eight significantly differential metabolites in serumfor the diagnosis of missed abortion. (B) ROC curves of the top eight significantly differential metabolites in villous tissue for the diagnosis of missed abortion. (C) ROC curve of the combined diagnostic model based on eight differential metabolites in serum. (D) ROC curve of the combined diagnostic model based on eight differential metabolites in villous tissue. The area under the curve (AUC) together with 95% confidence intervals were used to evaluate the diagnostic efficacy of each metabolite, and the diagonal line represented the random reference line. ROC curve analysis was performed on the top eight differentially expressed metabolites ranked by FDR in villous tissues. Each single metabolite exhibited moderate diagnostic efficacy for distinguishing missed abortion, among which C10:1 (AUC = 0.821) and C18 (AUC = 0.808) showed relatively superior performance. The combined diagnostic model established based on these eight metabolites yielded an AUC value of 0.885 (95% CI: 0.820–0.951), which was significantly higher than that of any individual metabolite. These results suggest that the combination of acylcarnitine metabolites in villous tissues can serve as potential auxiliary indicators for differentiating missed abortion ( Table 7 ; Figures 2B, D ). AUCs and 95% CI(Villous tissue).

Recent metabolomic studies demonstrated that serum metabolites including amino acids and acylcarnitines are significantly altered in early pregnancy complications such as missed abortion, providing potential biomarkers and insight into metabolic dysregulation mechanisms. For example, Xia et al. used LC-MS to identify differential serum metabolites associated with missed miscarriage ( 14 ) and Zhou et al. applied NMR-based metabolomics to characterize distinct metabolic profiles in missed abortion serum ( 13 ). Additionally, longitudinal pregnancy metabolomics has shown dynamic changes in amino acids and acylcarnitines during healthy gestation ( 15 ), and first-trimester serum acylcarnitines have been linked to other pregnancy metabolic outcomes ( 16 ). These findings support the emerging role of altered metabolite pathways in adverse early pregnancy outcomes. This study conducted targeted metabolomics analysis of serum and villous tissues from women with missed abortion and normal early pregnancy, the findings showed that missed abortion was associated with differences in 17 metabolites in serum samples and 25 metabolites in villous tissue samples from patients in early pregnancy. Firth regression analysis of serum samples revealed that eight amino acids, namely ORN, TYR, LEU+ILE+ProOH, ALA, VAL, PRO, CIT, ARG, were independently associated with an increased risk of missed abortion, acting as independent risk factors. Amino acid metabolism is critical for early embryonic development, supporting trophoblast proliferation, immune tolerance, and energy supply ( 17 ). The elevation of circulating amino acids in missed abortion may reflect maternal metabolic dyshomeostasis, potentially impairing early pregnancy maintenance. Villous tissue analysis showed that C5DC/C6OH, C6, C3DC/C4OH, CIT and ARG were risk factors for missed abortion, while C3, C0, C5, C4 and C4DC/C5OH served as protective factors. The carnitine pool plays a vital role in maintaining the body’s energy balance and overall health. Carnitine finely regulates the metabolic pathway through fatty acid transport ( 18 ). In this study, the reduction of free short-chain carnitines and the accumulation of medium- and long-chain acylcarnitines in villous tissues from patients with missed abortion indicate impaired β-oxidation, which may lead to energy deficiency, lipotoxicity, and impaired trophoblast viability and differentiation, ultimately resulting in embryonic developmental arrest and missed abortion. Additionally, ARG and CIT were significantly upregulated in villous tissues, consistent with their roles in nitric oxide synthesis and placental angiogenesis ( 19 ), their dysregulation may disrupt vascular development and maternal–fetal blood flow. Notably, only CIT was significantly upregulated in both serum and villous tissues, indicating its potential as a key shared metabolic mediator in missed abortion. CIT is involved in urea cycle and nitric oxide metabolism ( 20 ), and its concurrent elevation in serum and villous tissues indicates a coordinated dysregulation of nitrogen metabolism, which may impair maternal metabolic homeostasis and villous angiogenesis. CIT is an α-amino acid generated intracellularly from glutamine metabolism ( 21 ); it bypasses hepatic metabolism, enters the systemic circulation, reaches the kidneys, and is converted to arginine, through which it exerts diverse biological effects ( 22 ). In the present study, CIT levels were elevated in both the serum and villous tissue of patients with missed abortion. This contrasts with the generally reported protective role of CIT in various diseases, including cardiovascular and metabolic disorders, and suggests that increased CIT may raise the risk of missed abortion. However, the majority of differential metabolites were tissue-specific, with no highly consistent metabolites identified between serum and villous tissues. This discrepancy is biologically reasonable: serum reflects systemic maternal metabolism, while villous tissues directly mirror local embryonic and placental metabolic status. Differences in cellular composition, physiological function, and regulatory pathways between the two matrices inherently lead to distinct metabolic profiles. Furthermore, this study employed a targeted metabolomics approach with limited metabolite coverage, which may also contribute to the lack of overlapping differential metabolites. ROC curve analysis demonstrated that ORN, C6, and TYR exhibited the highest diagnostic efficacy in serum, while C10:1 and C18 performed best in villous tissues. The combined models of the top eight FDR-ranked metabolites achieved excellent discriminatory performance, with AUC values of 0.886 (serum) and 0.885 (villous tissue), significantly outperforming individual metabolites. These results indicate that both serum amino acid and villous acylcarnitine panels have potential as auxiliary discriminative indicators for missed abortion. This study has several limitations. First, gestational age was not well-matched between groups, with longer amenorrhea days in the missed abortion group, which may confound metabolic profile comparisons. Second, the sample size was moderate and from a single center, limiting generalizability. Third, targeted metabolomics only detected a predefined set of metabolites, potentially missing key regulatory molecules. Future studies should enroll larger, gestational age-matched cohorts, integrate untargeted metabolomics and multi-omics data, and validate key metabolites in independent cohorts to further elucidate the metabolic mechanisms of missed abortion. In conclusion, this study confirmed that missed abortion is characterized by serum amino acid hypermetabolism and villous acylcarnitine disorder, reflecting disrupted maternal–fetal metabolic crosstalk. The consistent alteration of CIT in both matrices highlights its potential as a core metabolic indicator. These findings advance our understanding of missed abortion pathogenesis.

This study performed targeted metabolomics profiling of serum and villous tissues from 78 patients with missed abortion and 75 women with normal early pregnancy undergoing elective termination, to characterize metabolic signatures associated with missed abortion. ROC analysis showed that ORN, C6 and TYR had the highest diagnostic efficacy in serum, while C10:1 and C18 performed best in villous tissue. The combined diagnostic model constructed from the top eight FDR-ranked serum metabolites (ORN, TYR, LEU+ILE+ProOH, ALA, VAL, PRO, CIT, ARG) achieved an AUC of 0.886, significantly outperforming any single metabolite, indicating good auxiliary diagnostic value for missed abortion. The combined model based on the top eight FDR-ranked villous tissue metabolites (C16OH, C5DC/C6OH, C10:1, C14OH, C18, C18:2, C12, C14) yielded an AUC of 0.885, with discriminatory ability superior to individual metabolites, suggesting that the villous acylcarnitine panel may serve as a potential auxiliary discriminative indicator for missed abortion. Notably, only CIT was consistently and significantly upregulated in both serum and villous tissue, while other differential metabolites were tissue-specific. The lack of highly consistent metabolites between the two sample types may be related to the limited coverage of the targeted metabolomics assay. These findings provide new insights into the metabolic pathogenesis of missed abortion.

This study involved screening patients with missed abortions who met the inclusion criteria and were treated at the Northwest Women’s and Children’s Hospital from March 2024 to December 2024. A total of 78 cases were included in the experimental group, while 75 cases of normal early pregnancy requiring termination were included in the control group. Patients with early missed abortion who met the diagnostic criteria in the 2020 Expert Consensus on the Treatment of Early Missed Abortion were selected. The inclusion criteria were as follows (1): Ultrasound showing a crown-rump length (CRL) ≥ 7 mm without fetal heartbeat (2); ≥ 25 mm average diameter of the gestational sac in the uterine cavity without an embryo (3); No yolk sac observed in the uterine cavity during pregnancy, and no embryo or fetal heartbeat detected even after 2 weeks (4); A yolk sac visible in the uterine cavity during pregnancy, but no fetal heartbeat observed even after 11 days. The tandem mass spectrometry system used for sample detection was the LC/MS/MS System (triple quadrupole mass spectrometer) from AB Sciex (model: API3200MD). The employed reagent kits included the Succinylacetone and Non-derivatized Multiple Amino Acids and Carnitine Detection Kit from Guangzhou Fenghua Biotechnology, among others. Statistical analyses were performed using SPSS 26.0 and R 4.2.1. Continuous variables are expressed as mean ± SD (normal distribution) or median (IQR) (non-normal distribution); categorical variables as n (%).Baseline characteristics between groups were compared using independent samples t-test or Mann-Whitney U test for continuous data, and chi-squared or Fisher’s exact test for categorical data, as appropriate.Differential metabolite levels were analyzed via Mann-Whitney U test, with false discovery rate (FDR) correction applied for multiple comparisons; FDR-adjusted P < 0.05 was considered significant.Firth penalized logistic regression was used to identify independent risk factors for missed abortion to address complete data separation issues. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic performance of individual metabolites and combined models, with the area under the curve (AUC) and 95% confidence intervals (CI) calculated. All tests were two-tailed, and P < 0.05 was considered statistically significant. Inclusion Criteria: a) Patients with missed abortion at ≤ 12 weeks of gestation; all patients received medical abortion with mifepristone combined with misoprostol and achieved villus expulsion. Those who failed to expel villi medically underwent artificial abortion to obtain villus samples. b) Diagnosis consistent with the relevant diagnostic criteria specified in the 2020 Expert Consensus on the Treatment of Early Missed Abortion. c) All enrolled patients met the safety criteria for medication use, with no drug allergy or contraindications, and normal liver and renal function. d) Age ranged from 20 to 35 years old. e) Patients had normal mental status and intact verbal communication and comprehension ability. f) All participants were fully informed of the research content, volunteered to participate, and signed the informed consent form. Exclusion Criteria: a) Patients with allergy or contraindications to prostaglandin drugs. b) Patients with a history of allergy or contraindications to mifepristone, such as those with endocrine diseases including adrenal diseases and diabetes. c) Patients with cardiac, hepatic, renal diseases or adrenal insufficiency. d) Individuals with a history of hematological diseases or hereditary porphyria. e) Anemic patients with hemoglobin concentration < 75 g/L indicated by routine blood examination. f) Patients diagnosed with or suspected of ectopic pregnancy, intrauterine device pregnancy, incomplete abortion, inevitable abortion, dilated cervical os, or other special abnormal pregnancy conditions. g) Cases lost to follow-up, with incomplete clinical data, or those with unevaluable therapeutic efficacy.