The performance of cortisol immunoassays is diminished by allo-THF, 11-deoxyF, 21-deoxyF, 6β-OHF and synthetic glucocorticoids . Therefore, the LC-MS-based profiling was conducted to generate serum cortisol signatures of SGA at 10 ~ 14 gestational weeks and quantify individual glucocorticoid levels. Based on the individual quantities, the metabolic ratios of precursors to their corresponding metabolites were also determined for indirect assessment of comparative enzyme activities.
Progesterone slowly increases from week 9 until week 32 of pregnancy, whereas the serum cortisol level increases significantly in the first trimester and peaks in the second week, followed by a decline in the third trimester . Biologically active free cortisol is generally decreased by the increased corticosteroid-binding globulin; however, in pregnant women the cortisol is replaced by a large quantity of progesterone . Maternal stress is one of the major risk factors for spontaneous abortion during the earliest gestational stages and the increased levels of maternal cortisol are correlated with a higher risk of miscarriage during pregnancy . However, the serum levels of cortisol in the SGA group at early stage of the first trimester were not statistically significant (P = 0.337; Fig. 1), consistent with previous studies involving women in the first and third trimesters [12, 13].
However, the metabolic signatures of cortisol, in this study, showed remarkable changes in SGA (Fig. 1). Among those signatures, 11β-HSD2 metabolizes cortisol to inactive cortisone, which is reactivated by 11β-HSD1. Both cortisol and cortisone are then reduced to dihydro-, tetrahydro- and allo-tetrahydrometabolites catalyzed by different reductases. In addition to antenatal glucocorticoid treatment , the decreased activity of placental 11β-HSD2 is caused by higher levels of maternal cortisol [5, 6], which also results in birth weight reduction . However, the maternal blood levels of cortisone and its 5β-reduced metabolite THE remained unchanged in SGA and non-SGA groups (Fig. 1). The metabolic ratios of E/F, indicating 11β-HSD2 activity, and THE/E were also unchanged (data not shown). In contrast, the metabolite of cortisol catalyzed by 5β-reductases, THF, was significantly increased (Fig. 1), while its metabolic ratio relative to cortisol, THF/F, was also higher in women carrying SGA fetus (Fig. 2A), similar to previous findings in pregnant women in third trimester . Our study demonstrated that these changes may also represent predictive biomarkers in the first trimester. The level of 5α-reduced metabolite of cortisol, allo-THF, tended to increase in the SGA group (Fig. 1), while its metabolic ratio relative to cortisol (allo-THF/F) was not significant (data not shown). Another reduced metabolite of cortisol monitored in this study, 20α-DHF, which is catalyzed by 20α-reductase, was also not significant between the groups (Fig. 1).
Cortisol is derived from two different precursors, 11-deoxyF and 21-deoxyF, catalyzed by 11β- and 21-hydroxylases, which are key enzymes in cortisol biosynthesis (Fig. 1). In contrast to suppression of cortisol biosynthesis under low 11-deoxyF levels in cord blood obtained from very low birth weight babies , maternal 11-deoxyF was decreased (P = 0.098, Fig. 1), which may be comparable to the apparent decrease in intrauterine fetal death and anencephalic fetus . The maternal F/11-deoxyF metabolic ratio was increased (Fig. 2B), indicating activated 11β-hydroxylase in cortisol synthesis, whereas a reduced 11β-hydroxylase was found in low birth weight neonates . The 21-Hydroxylase deficiency results in increased 21-deoxyF, which represents an alternative diagnostic marker in late-onset congenital adrenal hyperplasia , with similar activity between preterm and term infants .
The present study shows a significantly increased metabolic ratio of cortisol to 21-deoxyF in women carrying SGA fetus (P < 0.0003, Fig. 2C) with a remarkable decrease in serum levels of 21-deoxyF (P < 0.0001, Fig. 1). Both cortisol precursors are generally converted from 17α- and 11β-hydroxyprogesterones (17α-OHP and 11β-OHP), respectively. As 21-deoxyF may also be alternatively catalyzed by 11β-hydroxylase from 17α-OHP directly , we compared and found decreased metabolic ratios of 11-deoxyF/17α-OHP (P = 0.058) and 21-deoxyF/17α-OHP (P < 0.0005) in the SGA group (data not shown). Altered maternal serum levels of 11-deoxyF and 21-deoxyF may not only be derived from placenta, which lacks 17α- and 21-hydroxylase activities, but also from fetal adrenal glands entirely . The maternal 11-DeoxyF level generally increases with progressive pregnancy and peaks at 39 weeks , whereas 21-deoxyF is not elevated in premature infants . However, competitive metabolic pathways to produce 11- and 21-deoxyF were not identified because of many outliers.
The activity of 11β-HSD isoenzymes and its metabolic capacity for glucocorticoids play a pivotal role in regulating fetal growth. The placental mRNA level of 11β-HSD 1 is significantly increased during the late gestation period (38 ~ 40 weeks), while the levels of 11β-HSD 2 are decreased, corresponding to fetal maturation and labor . In contrast, pregnant women in their first trimester showed a compartmental distribution of 11β-HSD 1 and 2 at the feto-maternal interface, both of which were upregulated to possibly coordinate the interaction between isoenzymes . Excessive glucocorticoid levels inhibit fetal growth and are expressed by increased cortisol metabolite THF and the THF/F ratio [13, 28]. The finding was in accordance with the negative association of maternal serum THF levels with birth weight of babies (Fig. 4).
To the best of our knowledge, this is the first study to evaluate metabolic signatures of cortisol in the first trimester. Although the present work was designed to provide detailed information regarding cortisol metabolism in serum obtained from pregnant women with SGA fetus, this prospective cohort study has several limitations. First, the incidence of adverse fetal outcomes may reflect placental and maternal abnormalities, but the cortisol signatures in placenta were not measured in pregnant women in their first trimester. The quantitative results from mothers could be indirectly evaluated based on those obtained from babies due to protective mechanism against excessive glucocorticoid levels. However, no comparative experiment was performed. Third, the longitudinal data related to metabolic changes during pregnancy in this study were intended to identify metabolic changes in early stages.
In summary, a decreased serum 21-deoxyF combined with increased F/21-deoxyF combined with higher THF/F ratio, indicating fetal growth inhibition [13, 28], represents a potentially reliable biomarker for predicting SGA in the first trimester (Fig. 3). Studies are needed to investigate the molecular mechanism of enzymes related to cortisol metabolism and their association with lipid profiles.