Altered placental tryptophan metabolic pathway in human fetal growth restriction
Introduction
During pregnancy the essential amino acid tryptophan is actively transported to the fetus by the placenta [1]. Besides being utilised for protein synthesis by the placenta and fetus, another fate of tryptophan is degradation via the serotonin and kynurenine pathways. Tryptophan hydroxylase-1 is known to be highly expressed in the placenta of several species [2], and in mice placental synthesis of serotonin is important for early brain development [3]. Serotonin may also be important for maintaining vasodilation of the uterine circulation in the vicinity of the implanted placenta [4]. Tryptophan is also oxidised by enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), both of which are expressed in the human placenta (2). In early murine pregnancy IDO activity has been implicated in suppression of the maternal immune response to the conceptus [5], [6]. The role of this pathway in human pregnancy during early and late gestation is unclear.
Kynurenine produced as a result of either IDO or TDO activity is further degraded by vitamin B2- and B6-dependent enzymes to kynurenic acid (KA), anthranilic acid (AA), 3-hydroxykynurenine (HK), xanthurenic acid (XA) and 3-hydroxyanthranilic acid (HAA) (2, 6). IDO expression is induced by several inflammatory mediators, including interferon-γ (5, 7), resulting in decreased blood concentrations of tryptophan and increased blood concentrations of kynurenine and kynurenine pathway metabolites (5, 7). This is relevant to pregnancy because many kynurenine metabolites have actions within the central nervous system, and might impact on the fetal brain. For example, kynurenine is a convulsant [7], whereas kynurenic acid, a NMDA receptor antagonist, has anti-convulsant properties [8]. Quinolinic acid, a NMDA receptor agonist and an excitotoxin, has been implicated in many neurological disorders such as Alzheimer's and Huntington's disease [9], [10]. In addition, 3-hydroxykunurenine, anthranilic acid and 3-hydroxyanthranilic acid have cytotoxic properties [11], and picolinic acid is both a chemo-attractant for macrophages and can potentiate free radical damage [12], [13].
It has been proposed that placenta-derived kynurenine metabolites may be involved in the aetiology of perinatal brain damage (2). The human placenta expresses mRNAs of many kynurenine pathway enzymes throughout pregnancy, including both IDO and TDO [14], [15]. Placental explants produce tryptophan metabolites, including kynurenine, kynurenic acid, 3-hydroxyanthranilic acid, picolinic acid and quinolinic acid; all of these kynurenine pathway metabolites are also present in umbilical cord blood at term [15], [16]. Placental expression of several kynurenine pathway enzymes is increased in response to maternal infection, and placental kynurenine and quinolinic acid production increase with exposure to inflammation provoked by infection [15], confirming that activity of the kynurenine pathway in the placenta is very sensitive to infection and inflammation.
Little is known about circulating levels of tryptophan metabolites throughout normal physiological events such as pregnancy and labour, or of the ability of the placenta to produce kynurenines during stress, such as the hypoxia that often arises n late gestation and at parturition. Also, fetal growth restriction (FGR), which affects 5–15% of pregnancies in first world countries and up to 55% in developing countries, is a significant pregnancy disorder that has major consequences for the fetus and neonate such as higher rates of perinatal mortality, hypoxic ischemic encephalopathy, cerebral palsy, and a long-term morbidity extending into adulthood. Impaired placental function such as reduced activity of placental nutrient transporters contributes to the etiology of FGR [17], [18], but the involvement of altered tryptophan catabolism as a condition associated with human FGR is unclear.
Given that IDO gene expression is increased by inflammatory mediators [1], [2], [15], we hypothesized that activity of the tryptophan-kynurenine pathway would increase across gestation. Furthermore, because IDO is an oxygen-dependent enzyme and FGR is associated with fetal hypoxia, we hypothesized that kynurenine and the downstream metabolites would be decreased in pregnancies where FGR develops in late pregnancy. To examine these hypotheses we collected maternal and cord venous bloods from women undergoing delivery by either spontaneous or elective caesarean delivery at term. Furthermore, we collected placentas from women delivering at term, and compared these with samples collected at 1st trimester from elective termination of pregnancy. Finally, placental samples were also available to us from idiopathic FGR-affected and gestation age-matched pregnancies, allowing us to determine the effect of constitutive growth restriction on the expression of key kynurenine pathway enzymes in these placentas. Our results show that the tryptophan-kynurenine pathway is active in the placenta from early pregnancy, and that activity of the kynurenine enzymes are sensitive to condition that produce feto-placental growth restriction.
Section snippets
Materials and methods
Approval for this project was granted by the Monash Medical Centre Human Research and Ethics committee and from the Royal Women's Hospital Human and Research Ethics Committees, Melbourne with written consent given by all patients.
Maternal and umbilical cord blood
Arterial and venous cord blood was collected at the time of delivery from pregnancies of 37–41 weeks after spontaneous vaginal delivery (SVD, n = 10) or and caesarean section (C/S, n = 11). Blood samples were centrifuged at 3000 g and the plasma collected and stored at −20 °C until analysis.
Kynurenine pathway metabolites in cord blood
Tryptophan and all kynurenine metabolites were detectable in all maternal and cord arterial and venous bloods at term (Table 3). There were no consistent differences between arterial and venous cord samples for either mode of delivery, except for picolinic acid which was 3.9-fold higher in umbilical artery (4.3 μM) compared to vein samples (1.1 μM) after spontaneous vaginal delivery. Vaginal delivery was associated with higher kynurenine and 3-hydoxyanthranilic acid in arterial and venous cord
Discussion
This study shows that both the mRNAs and proteins of IDO and TDO are expressed in the human placenta from early gestation. Although these results cannot be taken as indicative of enzyme activity (the samples available to us were not suitable for this purpose), they do confirm that the genes products are translated through to cellular protein.
IDO is induced by pro-inflammatory signals, in particular by IFN-gamma, which may be involved in the local depletion of tryptophan, thereby suppressing of
Conclusion
Our study demonstrates that temporal expression of the key enzymes IDO and TDO may contribute to the pathogenesis of human FGR. Furthermore, early expression and activity of tryptophan metabolites, specifically the kynurenine metabolites in response to change in oxygen tension highlights their importance to feto-placental growth in human FGR.
Conflict of interest
Authors have no conflict of interests to declare.
Acknowledgements
The authors would like to thank Dr Ursula Manuelpillai and Dr Poonam Dharane from the Department of Obstetrics and Gynaecology, Monash University for their technical help. Monash Health is supported by the Victorian Government's Operational Infrastructure Support Program. The authors would like to thank the women who consented to provide samples to the study. The following are gratefully acknowledged: the clinical Research Midwives for sample collection and the Obstetrics and Midwifery staff of
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