Although over 90% of fetal growth occurs in the second half of gestation, maternal metabolism in the first trimester undergoes extensive changes in lipid storage, nitrogen species excretion and other metabolic pathways in order to facilitate fetal development . Thus, early maternal metabolic abnormalities could indicate, or even cause, abnormal implantation, fetal growth impairment or other adverse birth outcomes, before clinical symptoms appear. Using a 1H NMR-based metabolic profiling approach, we found early (end of first trimester) differences in urinary metabolic phenotypes in the pregnant women in the Rhea cohort study in whom PB and FGR subsequently occurred. These potentially predictive metabolic signatures of birth outcomes were correlated with aspects of metabolic syndrome. Furthermore, we observed a distinction between the metabolic signature of ‘medically indicated’/induced and ‘non-indicated’/spontaneous PB, suggesting a range of aetiological metabolic factors contributing to PB.
Despite the relative small number of induced preterm pregnancies (n = 28), a significant increase in N-acetyl glycoprotein fragments was observed in these women. The N-acetyl proton resonances arising at δ 2.04 ppm are frequently associated with inflammation-induced acute phase proteins such as alpha-1 glycoprotein when reported in serum , but the urinary source is less certain. One candidate is uromodulin, also called the Tamm-Horsfall glycoprotein, which is the most abundant protein found in urine . The N-acetyl glycoprotein resonance was positively correlated in this study with BMI. In the Rhea cohort, pregnant women with metabolic syndrome (and in particular the obesity component) had a high risk of encountering IPB (RR = 5.13, 95% Cl 1.97 to 13.38). The mechanisms relating N-acetyl glycoproteins to obesity and IPB, remain unclear; however, it is widely reported that high levels of adipose tissue can lead to systemic inflammation through release of cytokines such as interleukin-6, which could lead to an acute phase response . Higher N-acetyl glycoprotein levels in NMR spectra of women with PB were also found in a study profiling maternal serum and cord blood at birth . Spontaneous PB was specifically associated with higher urinary lysine, an essential amino acid that is limiting for growth, and is elevated in the plasma of premature infants . The steroid conjugate at 0.63 ppm, possibly arising from a soluble metabolite of pregnanediol, was also increased in SPB cases by 19%. This signal has also been identified in previous studies that detected it in the urine of second trimester pregnant women with subsequent fetal malformation and trisomy 21 [20, 44]. In our study, this steroid was also positively correlated with cholesterol and LDL-C, known sources for progesterone synthesis by the placenta.
With the exception of formate, a different metabolic profile (decreased urinary acetate, citrate, formate, glycine, tyrosine and trimethylamine) was associated specifically with poorer fetal growth. FGR remains difficult to assign owing to healthy biological variability in human size, hence in this study we used a definition based on customised birth-weight percentiles designed to better differentiate between infants who are small because of restricted growth and infants who are small but have reached their likely individual growth potential (see Methods) [18, 26]. A similar pattern of associations was observed for this parameter as for the more conventional classification of SGA; however, FGR resulted in more statistically significant associations because of larger sample size. The FGR metabolite profile was broadly inversely associated with plasma insulin and positively correlated with HDL-C levels. Of these metabolites, formate, tyrosine and trimethylamine were all found to be significantly positively correlated with each other, suggesting a common source of variation (ρ(formate-tyrosine) = 0.38, ρ(formate-trimethylamine) = 0.21 and ρ(tyrosine-trimethylamine) = 0.26). Elevations of several of these metabolites in blood have been previously associated with risk of insulin resistance [45, 46]; however, the biological significance of low urinary levels of these molecules is less clear. Low urinary formate has been previously associated with increased hypertension in a large multinational study  and interestingly, hypertension in the first trimester of pregnancy was the most significant risk factor for PB and FGR in the Rhea cohort . However, the association between formate and BP observed was not statistically significant in our study cohort. Several of the metabolites in the FGR signature (acetate, formate, tyrosine, trimethylamine) are known to be consumed or produced in significant quantities by gut microbes [48–51], hence the association might reflect a specific gut microbial distribution or a dietary pattern that selects for such a distribution. A recent study reported dramatic change in gut microbial composition throughout pregnancy causing increased insulin resistance and greater adiposity; although normally associated with disease this may be of benefit during pregnancy . This indicates that the composition of gut microbiota in pregnant women could influence their metabolic homeostasis and their pregnancy outcomes. Daily intake of 5 mg of supplemental folic acid in the whole Rhea population (n = 1,279) was associated with a 66% decrease in the risk of delivering an SGA neonate (RR = 0.34; 95% CI 0.16 to 0.73) . However, formate levels were not correlated with supplementary folate intake in our study population (ρ = −0.05 and P = 0.23).
Despite our study not being directly comparable with previous metabolomics studies investigating birth outcomes, owing to differences in the analytical platform used and the biofluids studied (often cord blood serum or amniotic fluid), and because our samples were taken at the end of the first trimester (whereas most previous metabolomics studies have examined late pregnancy samples), some similarities with other investigations were observed. In addition to the instances cited above, a previous study in women with subsequent FGR also reported decreased levels of urinary trimethylamine, tyrosine and glycine . However, many more metabolomic studies have focused on events during pregnancy such as pre-eclampsia rather than birth outcomes; in our work, we excluded women with pre-eclampsia, making comparison with these inappropriate.
Our study has a number of other important limitations. Firstly, our study was not specifically designed to examine FGR, and only a limited number of these cases were available within our dataset. Secondly, although our study is unique in defining associations between metabolism during early pregnancy and birth outcomes, it is not possible at this stage to distinguish between pregnancy-induced effects and underlying metabolic risk factors. However, this does not negate the potential value of urinary metabolites in general as biomarkers of risk of negative birth outcomes, and our exploratory study has generated several hypotheses for future investigation. It is also possible that our observations reflect aetiological factors specific to the Rhea cohort, which experience an abnormally high rate of PB, and are not generalisable to the broader European population. Specimens from an independent cohort would be needed to validate our findings, and several efforts to complete comparable studies in large birth cohorts are currently underway, such as the Human Early-Life Exposome (HELIX) project . This consortium aims to implement novel exposure assessment and biomarker methods (including metabolomics) to characterise the total exposure from conception to multiple environmental factors (the exposome [55–57]) and associate these with child health outcomes. Applied as an untargeted approach, metabolomics captures information about endogenous metabolism and exogenous exposures simultaneously, making it in principle an ideal tool for exploring the exposome.