Untargeted metabolomics reveals key pathways in miscarriage: steroid, folate, fatty acid & glycosaminoglycan metabolism

Abstract

Metabolites influencing miscarriage outcomes remain understudied. We hypothesized that aberrant metabolism impacts threatened miscarriage outcomes and that understanding these pathways could offer new management strategies. This case-control study analyzed serum metabolomics from 80 women between 5 and 12 weeks' gestation at KK Women's and Children's Hospital, Singapore, comparing three groups: women with threatened miscarriage who miscarried (TM_MC), those with ongoing pregnancies (TM_O), and women with normal pregnancies (NP). Using untargeted liquid chromatography-mass spectrometry and pathway analysis through MetaboAnalyst 5.0 and the Kyoto Encyclopedia of Genes and Genomes, 267 metabolites across 12 enriched pathways were identified. Dysregulations in steroid (AUC 0.82), folate (AUC 0.59), fatty acid (AUC 0.70), and glucosaminoglycan (AUC 0.64) pathways distinguished women who miscarried from those with ongoing pregnancies (TM_MC vs TM_O). We provide initial insights into the metabolic profile associated with miscarriage, highlighting disruptions in steroid hormone, fatty acid, folate, and glucosaminoglycan biosynthesis. Further validation may support biomarker development for prognostication.

Keywords: Reproductive biology; Reproductive disorders.

Fig. 1. Partial least squares-discriminant analyses using serum metabolites of women with normal pregnancies and threatened miscarriages.

a The partial least squares-discriminant analysis (PLS-DA) of serum metabolites in patients with threatened miscarriage (TM) vs those with normal pregnancies (NP), with the PLSDA showing separation into 2 distinct clusters (5 fold cross-validation with 5 principal components – R² = 0.99, Q² = 0.65, classification accuracy = 0.99). b The PLS-DA of metabolites in patients with threatened miscarriage who eventually miscarried (TM_MC) vs those who had ongoing pregnancies (TM_O). PLSDA shows poor separation (5 fold cross-validation with 5 principal components – R² = 0.98, Q² = 0.22, classification accuracy = 0.66).

Fig. 2. Volcano plots of metabolites of women with normal pregnancies and threatened miscarriages.

a Volcano plot for metabolites in patients with threatened miscarriage (TM) vs those with normal pregnancies (NP). Metabolites which were significantly different (Fold Change (FC) ≥ 2 or ≤0.5, and raw p-value < 0.05) are highlighted. FC is expressed with NP as reference. b Volcano plot for metabolites in patients with threatened miscarriage who eventually miscarried (TM_MC) vs those who had ongoing pregnancies (TM_O). Metabolites which were significantly different (Fold Change (FC) ≥ 2 or ≤0.5, and raw p-value < 0.05) are highlighted. (FC is expressed with NP as reference). Red hues indicate metabolites with increased abundance in the TM group compared to NP (positive fold change), while blue hues indicate metabolites with decreased abundance in the TM group compared to NP (negative fold change). Black/gray points represent metabolites that do not meet the statistical significance threshold.

Fig. 3. Box plots of significantly altered serum metabolites in women with normal pregnancies,threatened miscarriage who eventually miscarried, and threatened miscarriage with ongoing pregnancies.

Box plots showing the normalized concentrations of (a) estrone sulfate, (b) dermatan, (c) 4α-hydroxytetrahydrobiopterin and (d) palmitic acid in the NP (red), TM_MC (green), and TM_O (blue) groups. The yellow diamonds represent the mean concentrations for each group, while the black dots indicate individual data points. NP normal pregnancies, TM_MC patients with threatened miscarriage who eventually miscarried, TM_O patients with threatened miscarriage who had ongoing pregnancies.