The relationship between the placental serotonin pathway and fetal growth restriction

Biochimie 161 (2019) 80e87

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The relationship between the placental serotonin pathway and fetal growth restriction Suveena Ranzil a, b, David W. Walker c, Anthony J. Borg d, Euan M. Wallace a, b, Peter R. Ebeling e, Padma Murthi b, d, e, f, * a

Department of Obstetrics and Gynaecology, Monash University, Australia The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia RMIT University, Bundoora, Victoria, Australia d Department of Maternal-Fetal Medicine, Pregnancy Research Centre, Royal Women's Hospital, Parkville, Victoria, Australia e Department of Medicine, School of Clinical Sciences, Clayton, Victoria, Australia f Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Parkville, Victoria, Australia b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 June 2018 Accepted 26 December 2018 Available online 31 December 2018

Fetal growth restriction (FGR) is a complex disorder of human pregnancy that leads to poor health outcomes in offspring. These range from immediate risks such as perinatal morbidity and stillbirths, to long-term complications including severe neurodevelopmental problems. Despite its relatively high global prevalence, the aetiology of FGR and its complications is not currently well understood. We now know that serotonin (5-HT) is synthesised in the placenta and is crucial for early fetal forebrain development in mice. However, the contribution of a disrupted placental 5-HT synthetic pathway to the pathophysiology of placental insufficiency in FGR and its significant fetal neurodevelopmental complications are unclear. © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Fetal growth restriction (FGR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2.1. Definition and prevalence of FGR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2.2. Aetiology of FGR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2.3. Diagnosis of FGR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2.4. Management of FGR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 2.5. Complications of FGR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Placental insufficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 3.1. The placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 3.2. Placental structure and function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 3.3. Normal placental development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 3.4. Abnormal placental development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 3.5. Placental insufficiency associated with FGR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 3.6. Biomarkers of placental insufficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Placental tryptophan metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 4.1. Placental 5-HT synthetic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 4.2. Key functions of 5-HT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

* Corresponding author. Department of Medicine, School of Clinical Sciences and The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Victoria, Australia. E-mail address: [email protected] (P. Murthi). https://doi.org/10.1016/j.biochi.2018.12.016 0300-9084/© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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4.3. Consequences of abnormal 5-HT levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 The effects of placental insults on tryptophan metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 5.1. Inflammation and tryptophan metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 5.2. Hypoxia and tryptophan metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Nutritional factors and their regulation of the 5-HT synthetic pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

1. Introduction Fetal growth restriction (FGR) is an important pregnancy complication that has significant effects on neonatal morbidity and mortality, and the lifelong health outcomes of offspring [1]. Although the exact pathophysiological process behind FGR remains unclear, FGR is often associated with placental insufficiency [2], and a number of maternal, fetal and placental factors are believed to play contributing roles. Several placental insults, including placental hypoxia and inflammation [3,4], are proposed to contribute to placental insufficiency, leading to the altered expression and synthesis of certain growth factors, cytokines and transcription factors [4]. Given this apparent multifactorial nature, our laboratory is interested in understanding the contribution of an altered placental serotonin (5-HT) pathway to human FGR. The human placenta synthesizes 5-HT from tryptophan, which has several important functions in human pregnancy. In mice, there is evidence to suggest that abnormalities in placental 5-HT levels disrupts normal brain development [5]. If placental insufficiency in FGR alters tryptophan metabolism to 5-HT, this may result in fetal brain abnormalities. The changes in the placental serotonin pathway may also contribute to the development of FGR. Thus, altered placental 5-HT could be a cause or a consequence (or both) of FGR, depending on the timing of insult or dysfunction during pregnancy. However, the association between FGR and altered placental 5-HT synthesis and metabolism is yet to be fully uncovered.

metabolism [9] and multiple gestation pregnancies [11], whilst the most common uteroplacental factor is reduced placental perfusion, in which inadequate placentation leads to chronic placental insufficiency [2]. 2.3. Diagnosis of FGR The diagnosis of FGR and its aetiological basis is complex and involves a number of investigations. FGR is diagnosed through abnormalities in estimated fetal weight and potentially symmetry, amniotic fluid dynamics and/or fetal anatomy assessment [12]. An elevated head circumference to abdominal circumference ratio (HC/AC ratio 95th percentile) [13] and decreased amniotic fluid index (AFI <5 cm) [14] are commonly used indicators of additional pathology found in FGR. Measurements of Doppler velocimetry to identify placental insufficiency allow for simultaneous assessments of the maternal, placental and fetal circulations [12,15], and include umbilical artery Doppler, uterine artery Doppler and middle cerebral artery Doppler. Other Doppler velocities that may be used are cerebroplacental ratio (CPR), aortic isthmus Doppler and ductus venosus Doppler [12]. Following diagnosis, regular surveillance is performed using measurements such as umbilical artery Doppler velocimetry and the modified biophysical profile score [16]. 2.4. Management of FGR

2. Fetal growth restriction (FGR) 2.1. Definition and prevalence of FGR FGR is the second most common cause of perinatal mortality and affects 5e10% of all pregnancies. It is defined by the American College of Obstetrics and Gynecology as a sonographic estimated fetal weight of less than the 10th percentile for gestational age [1]. In order to separate FGR from otherwise healthy small for gestational age infants, additional pathologies like a reduced amniotic fluid index or growth asymmetry must be present [6]. Thus FGR is actually a syndrome characterised by failure of the fetus to reach its genetically predetermined intrauterine growth potential, and is influenced by a number of factors [1]. 2.2. Aetiology of FGR The aetiology of FGR is multifactorial and influenced by a number of maternal, fetal and uteroplacental factors, which may overlap. Maternal factors include clinical diseases like hypertensive disorders of pregnancy [7], nutritional disorders of chronic malnutrition and vitamin D deficiency [6], smoking and other drugs [8] and constitutional factors, specifically ethnicity, depression and stress levels [1]. Fetal factors include chromosomal abnormalities, genetic syndromes [9], intrauterine infections [10], inborn errors of

Presently no intrauterine management for FGR exists, and perinatal management involves balancing fetal and neonatal risks to identify the appropriate timing for iatrogenic preterm delivery [17]. No randomised trials have been conducted on elective delivery after diagnosis of FGR in term pregnancies. Available data on stillbirth risk suggest delivery occur as early as 37 weeks' gestation to reduce the stillbirth rate in this group [18], however there is a lack of consistent evidence to safely recommend a specific time of delivery. This is even more complex in preterm FGR presenting at less than 34 weeks’ gestation, as fetal health may decline more rapidly with increased morbidity associated in this subset when compared to fetuses exhibiting normal growth [19]. Alternatively, the results of a recent randomised control trial of delivery in preterm FGR suggest that optimal timing of delivery may be achieved through the use of monitoring with ductus venosus Doppler changes and cardiotocographic heart rate short term variation (c-CTG STV) [20]. 2.5. Complications of FGR The clinical consequences of FGR can be severe with both immediate and lifelong risks. There is an increased rate of stillbirths [21], with long-term health problems that include neurodevelopmental disorders due to poor brain development, as well as cardiovascular and endocrine diseases in adulthood [1].

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3. Placental insufficiency 3.1. The placenta The placenta is required for normal fetal growth and development as well as normal advancement of pregnancy. Although its development is tightly controlled and modulated throughout the gestational period [22], problems may arise, leading to placental pathology that can have detrimental impacts on both immediate and lifelong maternal and fetal health [23,24].

displaced until about the eleventh week of gestation [35]. There are advantages to this relatively hypoxic environment early in pregnancy, including limitation of damage to biomolecules from potentially harmful reactive oxygen species [36]. Placental development is a well co-ordinated process that occurs in a spatiotemporal manner, and as a result, developmental abnormalities can result in significant placental dysfunction and fetal and maternal health complications.

3.4. Abnormal placental development 3.2. Placental structure and function The human placenta on average is ~22 cm in diameter, 2.5 cm thick centrally and weighs roughly 500 g. It consists of the chorionic plate on the fetal side and the basal plate on the maternal side, adjacent to the maternal endometrium [25]. The main functional units are the chorionic villi, which have a thin membrane that separates fetal from maternal blood in the intervillous space [26]. It is across this 3-4 cell layer membrane that the placenta performs its vital functions. Apart from this separating membrane, exchange between the maternal and fetal circulations is further facilitated by a large placental surface area and remodelling of maternal uterine arteries for optimal perfusion [25,27]. The main function of placental exchange is to supply oxygen and nutrients to the fetus [25]. It is also involved in the removal of carbon dioxide and other waste products from the fetal circulation, metabolism of substances and release of products into the maternal and/or fetal circulations, protection of the fetus against xenobiotics, and synthesis and secretion of a number of steroid and peptide hormones that affect maternal and fetal metabolism, growth and other processes in pregnancy [22,25]. 3.3. Normal placental development Placental development continues throughout the gestational period, in order to meet the increasing metabolic needs of the growing fetus, and begins with implantation in early first trimester [25]. After fertilization, the single cell zygote rapidly divides to form the morula, which travels through the oviduct to the uterus, proliferating and differentiating into the blastocyst [28]. The blastocyst consists of approximately 50 cells and includes two layers; the inner cell mass, which eventually develops into the embryo and the outermost trophoblast layer, which later becomes the fetal component of the placenta [28]. Once implantation on the uterine wall occurs, the trophoblast cells further proliferate and differentiate along either the villous or extravillous pathway [29]. Mononuclear cytotrophoblasts in the villous pathway differentiate to form the multinucleated syncytiotrophoblast layer, an epithelial covering the surface of the chorionic villi [29]. It is this syncytium that comes into direct contact with the maternal circulation in the intervillous space, and allows the exchange of oxygen and nutrients [30]. The extravillous pathway generates columns of specialised invasive cytotrophoblasts which, by days 13e14 of pregnancy, begin to migrate and invade the decidua (maternal component of the placenta) and then continue to remodel the maternal spiral arterioles to the depth of the inner third of the myometrium [31e33]. This invasive transformation facilitates blood flow at a lowered arterial pressure, through increased dilatation and compliance of the spiral arterioles [34]. Early placental development occurs in a low oxygen environment (O2 concentration <20 mmHg). This is because maternal arterial inflow through the spiral arterioles is not fully established until late first trimester, given that the mouths of most vessels are initially plugged by invasive cytotrophoblasts, which is not

Placental insufficiency is thought to result from abnormal placental development and function, and is correlated with an altered production of placental growth factors and cytokines [37].

3.5. Placental insufficiency associated with FGR A major contributor to late-onset FGR is chronic placental insufficiency [2]. And since the metabolically active placenta extracts 40% incoming oxygen and 70% glucose supplied to the uterus, even mild placental dysfunction can reduce nutritional supply to the fetus and disrupt fetal growth [17]. Causes of sustained placental insufficiency are most often associated with either maternal or fetal vascular compromise. The most common of these is uteroplacental underperfusion as a result of maternal vascular disease, caused by insufficient trophoblast invasion of the spiral arterioles during placental development [2]. Abnormal trophoblast invasion, leading to inadequate remodelling of the spiral arterioles, is thought to create high resistance in the uterine arteries which decreases placental perfusion [1]. This abnormality is also considered to be a key component in the development of other pregnancy complications, like preeclampsia, miscarriages and preterm labour [38]. Reduced cytotrophoblast proliferation [39] and increased cytotrophoblast apoptosis [40] are believed to impair migration to the decidua and invasion of the spiral arterioles. These abnormalities also appear to impair villous cytotrophoblast fusion to form the syncytiotrophoblast layer, thereby reducing the syncytiotrophoblast mass available for nutrient and oxygen exchange, further limiting fetal growth in utero [3]. However, the underlying cause and molecular mechanisms of abnormal trophoblast invasion are yet to be elucidated [7]. In addition to the cytotrophoblastic dysfunction described above, various studies suggest that resultant hypoxia, oxidative stress or both may have an effect, through the generation of reactive oxygen species (ROS), activation of the complement cascade or inflammation [3,4]. However, Burton suggests that the placental pathophysiology behind FGR is not sufficient to generate oxidative stress as occurs in preeclampsia [41], and other studies indicate that the abnormal placenta in FGR may be an intermediate between normal and preeclampsia [7]. There is also evidence indicating that coagulability is disrupted in these pathological pregnancies, with increased fibrin deposition and thrombi in vessels [42]. Fig. 1 provides a simplified depiction of the adverse events during placental development that are currently thought to contribute to FGR. The pathophysiology behind FGR appears to be multifactorial, but the effects of a disrupted placental 5-HT synthetic pathway are unclear. 5-HT may have a potent role in the arterial contraction linked to hypertension [43] and placental dysfunction [44] and it is therefore possible that increased 5-HT production and/or receptor expression in FGR contributes to the disorder through effects on the endothelium.

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Fig. 1. Diagrammatic depiction of the pathophysiology behind placental insufficiency leading to FGR. Abnormal placental development, as a result of trophoblast and endothelial dysfunction, leads to underperfusion of the placenta and decreased syncyctiotrophoblast mass available for transfer of O2 and nutrients to the fetus. Placental insults believed to potentiate placental insufficiency include hypoxia, oxidative stress, inflammation and disrupted coagulability.

3.6. Biomarkers of placental insufficiency Each proposed contributory pathway is associated with specific molecular and genetic markers. For example, oxidative stress is marked by an increase in ROS [45], and inflammation is marked by an increase in pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFa), interferon gamma (IFNg) and interleukins IL-1a and IL-1b [46,47]. Tissue response to hypoxia can be measured through the upregulation of various biomarkers, such as hypoxia-inducible factors (HIF) and vascular endothelial growth factors (VEGF), as well as their regulating genes [48]. Measurements of mRNA expression of placental hypoxia markers show statistically significant upregulation of HIF-1a, VEGFA and VEGFR2 in term FGR placentae compared to controls. With a 3-, 5- and 6-fold increase respectively, this indicates that placental hypoxia is present in these pathological pregnancies [48]. Moreover, a recent rat study suggests that heme oxygenase-1 (HO-1) expression in placental villous explants exposed to chronic hypoxia is decreased compared to normoxic controls [49]. Deficiencies in HO-1 are thought to be associated with pregnancy disorders including FGR, however more work is required before definitive conclusions can be derived [50]. As this field is still advancing, it is impossible to pinpoint an exact pathophysiological basis for this pregnancy complication. Also unclear is the relationship between placental insufficiency in FGR and disrupted placental pathways during pregnancy, such as tryptophan metabolism to 5-HT.

4. Placental tryptophan metabolism Tryptophan is an essential amino acid that can only be obtained through the diet. Major dietary sources of tryptophan include oats, milk, tuna, bread, turkey, chicken and bananas, with the recommended daily intake in adults being between 250 and 425 mg/day [51]. This amino acid plays an important role in pregnancy. Maternal tryptophan is an essential factor in normal pregnancies, which is supported by an elevation in free maternal tryptophan during gestation [52]. Not only is this amino acid actively transported to the fetus via the placenta for protein synthesis for

growth and development, but tryptophan is also metabolised in the placenta via four pathways to produce functionally important metabolites [52]. A detailed explanation of each of these pathways is beyond the scope of this review, and focus will be placed on one of the two major pathways, leading to the production of 5-HT.

4.1. Placental 5-HT synthetic pathway 5-HT is a major product of tryptophan metabolism in the placenta, formed through the hydroxylation pathway [52]. Some of the 5-HT produced in the placenta is then converted into melatonin by the enzymes arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT) [53]. 5-HT is synthesised from its precursor L-tryptophan. The first step in this process is the hydroxylation of L-tryptophan to 5hydroxytryptophan (5-HTP) by the rate limiting enzyme tryptophan hydroxylase 1 or 2 (TPH1 or TPH2) [52]. TPH1 is required for 5-HT synthesis peripherally and is found various tissues including the placenta. This peripheral 5-HT synthetic pathway is responsible for the 5-HT found in the blood circulation [54]. On the other hand, TPH2 has traditionally been thought to be responsible for 5-HT synthesis in the brain and is located in the raphe nuclei neurons, as well as producing 5-HT in the enteric nervous system to promote gut motility [54,55]. However, a recent study has found that TPH2 is also expressed in human and mouse placentae [56]. The next step in the 5-HT synthesis process is the decarboxylation of 5-HTP by aromatic-L-amino acid decarboxylase (AADC or AAAD) to produce 5-HT [51]. Fig. 2 depicts the peripheral and central tryptophan metabolic pathways, with a focus on 5-HT synthesis. The other major peripheral tryptophan metabolic pathway produces kynurenine metabolites [57]. A landmark study by Bonnin et al. was the first to discover that mouse and human placentae are a source of 5-HT for the fetus [5]. Previously it was believed that maternal 5-HT was a major contributor to fetal 5-HT levels [59], despite no evidence of direct transfer of maternal 5-HT to the fetal circulation. Although the contribution of maternal 5-HT is unknown, it is now clear that the placenta is the major source of 5-HT for the fetal mouse for early forebrain development [5].

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Fig. 2. Diagram illustrating tryptophan metabolic pathways, with a focus on the peripheral and central 5-HT synthetic pathways. Adapted from Patrick and Ames 2015 [58].

Bonnin et al. directly tested placental synthesis of 5-HT in both mouse and human placentae, and neosynthesis of 5-HT was evidenced in both models. In mice, 5-HT synthesis occurred from embryonic day 10.5 (E10.5), and human 5-HT synthesis occurred at 11 weeks’ gestation. Furthermore, immunohistochemistry identified that TPH1 and AADC are expressed in the syncytiotrophoblast of mouse placentae, confirming that the placenta has the ability to synthesise 5-HT from tryptophan [5]. Wu et al. (2006) reported that in addition to syncytiotrophoblast, multiple sites including the visceral endoderm cells in the yolk sac of the mouse extraembryonic tissues may also contribute to fetal 5-HT production [60]. The 5-HT transporter (SERT) is thought to extract 5-HT from the maternal circulation for metabolism in the syncytiotrophoblast [61], in order to regulate 5-HT levels in the maternal circulation and moderate the vasoactive effects of 5-HT [43]. The expression of SERT and serotonergic receptors, specifically 5-HT2A receptor subtype has been identified in the trophoblast and fetal capillary endothelium of the human placenta [62e64]. 4.2. Key functions of 5-HT 5-HT is an important neurotransmitter that is well known for its effects on mood, sleep, cognition, memory, appetite, executive function, social behaviour and sensory gating [43,58,65]. Deficiency of this monoamine neurotransmitter is believed to partially explain the development of depressive illnesses [65]. In addition to being a neurotransmitter, 5-HT also regulates a number of key processes in neurodevelopment, including cell proliferation and neuronal differentiation, migration and synaptogenesis [66,67]. Several studies have also shown serotonergic innervation in the brain to be an integral process occurring during fetal brain development. Serotonergic neurons have been identified from embryonic days E11.5 to E12.5 in mice brains [68], and from the fifth week of gestation in human neural tissue [69]. In humans,

5-HT synthesis continues throughout gestation and peaks at five years of age, at levels approximately double that found in the adult brain [70], and then eventually declines to adult levels as brain maturation occurs [71]. The development and outgrowth of serotonin-producing neurons in rodents and humans is fairly similar, involving an early spurt of development and then a decline in levels towards adolescent years [72]. In rats, serotonergic neurons are initially found in two separate groups in the midline of the rhombencephalon [68]. They then elongate and divide into clearly defined fiber tracts, entering the medial forebrain bundle and reaching the hippocampus and basal forebrain by E16-E17 [73], eventually arriving at the frontal cortex by E20 [74]. In humans, the serotonergic neurons are initially present in the brainstem [69], appearing at the cortical plate by the thirteenth week of gestation [75]. They are found predominantly in the medial and dorsal raphe nuclei by at least 15 weeks’ gestation, sending projections to the cortex and hippocampus [76,77]. 5-HT synthesised in the placenta might also contribute to human fetal brain development. In the mature adult brain, peripherally synthesised 5-HT cannot cross the blood brain barrier, and therefore must be synthesised in the brain itself [54]. Tryptophan, on the other hand, is able to cross the blood brain barrier in adults, and the conversion of tryptophan to 5-HT in the brain occurs via the metabolic pathway involving TPH2. 5-HT concentrations in the adult brain therefore depend on circulating tryptophan levels [54]. Conversely, the immature fetal blood brain barrier enables exogenous 5-HT to pass into the brain [78]. This means that 5-HT synthesised peripherally in the placenta has the potential to modulate neuro-developmental processes in the fetal brain because several classes of 5-HT receptors are expressed throughout the developing brain, even before endogenous 5-HT axons are present [78,79]. In 2011, Bonnin et al. established the importance of the placental source of 5-HT for mouse fetal brain development [5]. Their findings demonstrate that the placenta is the main source of 5-HT for

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early fetal forebrain development in mice from E10.5e15.5, a period in which there is significant neurogenesis and axon growth. There is then a shift to dependence on an endogenous source, supplied by the axons of dorsal raphe neurons in the developing forebrain. Although placental 5-HT also contributes to fetal hindbrain development, the hindbrain mainly depends on an endogenous supply of 5-HT from dorsal raphe neurons throughout the gestational period [5]. However, a major limitation of these findings is that they apply specifically to an animal model of pregnancy and fetal brain development. Whilst Bonnin et al. suggest that E10.5e15.5 corresponds to the first and early second trimesters of human pregnancy, the contribution of placental 5-HT to human fetal brain development is currently unknown [5]. 4.3. Consequences of abnormal 5-HT levels Abnormalities in 5-HT levels in the brain have also been linked to neurodevelopmental disorders such as autism spectrum disorder (ASD) [54]. The brains of individuals with ASD have lower concentrations of 5-HT compared to those of controls [80]. Furthermore, 5-HT deficiency in neonate mice leads to abnormally large cortical brain growth and behavioural characteristics, similar to the changes found in individuals with ASD [81]. Additionally, the normal pre-pubertal peak in 5-HT synthesis, thought to promote neuronal growth and differentiation, is absent in children with ASD [70]. These findings suggest that ASD is associated with low 5-HT concentrations in the brain, although no underlying mechanism has been found. There is also evidence suggesting that gestational 5-HT dysfunction negatively affects fetal brain development, which may play a role in the pathogenesis of neurodevelopmental disorders. For example, SERT and 5-HT receptors are expressed early in the period of brain development [82,83], and polymorphisms in SERT has been shown to be connected to neurodevelopmental disorders such as schizophrenia and ASD in mice [84,85]. Moreover, abnormal 5-HT levels in the brain during development have been associated with the pathogenesis of numerous neurocognitive disorders in mice and rat models, including depression, anxiety, ASD, schizophrenia and Alzheimer's disease [79,86,87]. It is proposed that atypical 5-HT concentrations in the brain during development disrupt the formation of brain circuitry and thereby cause functional problems. For instance, in vitro and in vivo disruption of 5-HT signalling on axon guidance was demonstrated for thalamocortical axons resulting in abnormal axon trajectories in mice [88]. Both increased and decreased serotonergic activity are thought to lead to abnormal brain development in the fetus. For example, 5HT deficiency achieved using Pet1 knockout mice leads to a significant reduction in serotonergic neuron amount and differentiation, with evidence of behavioural abnormalities later in life [68]. Recent studies have also demonstrated that increased 5-HT activity in the brain may contribute to abnormal neuronal migration and cortical development [89e92]. Given the potentially severe consequences of 5-HT disruption on human fetal brain development, and the possible contribution of placental 5-HT to the developing brain, it is important to understand what factors affect placental 5-HT synthesis during this critical period. 5. The effects of placental insults on tryptophan metabolism The exact relationship between FGR and altered tryptophan metabolism in the placenta is unclear. Since no one specific placental insult is thought to contribute to placental insufficiency in utero, the results of in vitro studies can only suggest possible

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associations. Previous animal and human in vitro studies have provided evidence that inflammation and hypoxia affect placental tryptophan metabolism, as will be explored in this review, but it is beyond its scope to explore the effects of every potential placental insult in detail. 5.1. Inflammation and tryptophan metabolism There is evidence that above normal levels of maternal inflammation lead to abnormal brain development and poor developmental outcomes in offspring. Administration of different doses of the immunostimulant polyriboinosinic-polyribocytidylic acid [poly (I:C)], which increases inflammatory cytokines in the maternal circulation, appears to result in disturbed brain development and behavioural defects in offspring mice [93]. There is also evidence that increased maternal inflammation disrupts tryptophan metabolism in the placenta. With respect to the kynurenine pathway, Goeden et al. have demonstrated that mild maternal inflammation induced by poly (I:C) increases placental Ido1 gene expression in the placentae of pregnant mice [94]. A number of inflammatory mediators, such as TNF- a, IL-1b and IFN- g, are known to induce IDO expression during a systemic inflammatory response, thereby increasing tryptophan metabolism to kynurenine [95]. However, these findings only relate to animal models, and may not represent the effects of inflammation on the human placental kynurenine synthetic pathway. Goeden et al. have also demonstrated the effects of mild maternal inflammation on placental tryptophan metabolism to 5HT. Administration of poly (I:C) to pregnant dams led to a transient increase in tryptophan concentration in the placenta at E12, followed by upregulation of Tph1 gene expression and an increase in TPH1 enzymatic activity [94]. These alterations in the 5-HT synthetic pathway led to a sustained elevation in 5-HT output to the fetus, increasing 5-HT concentration in the rostral forebrain and decreasing serotonergic axon outgrowth in this region. Moreover, addition of the selective TPH inhibitor para-chlorophenylalanine (pCPA) prevented 5-HT accumulation in the brain and blunting of serotonergic axon outgrowth [94]. These statistically significant findings suggest that excessive maternal inflammation during human pregnancy may lead to increased 5-HT synthesis in the placenta and output to the fetus, resulting in abnormal serotonergic axon outgrowth in the developing forebrain. 5.2. Hypoxia and tryptophan metabolism Given that IDO is an enzyme that depends on oxygen for its function [96], it follows that its expression and activity may be downregulated in hypoxic conditions. The effects of placental hypoxia on kynurenine synthesis from tryptophan have been demonstrated in a recent ex vivo study by Murthi et al. [97]. In this study, first and third trimester human placental explants were exposed to hypoxic (5e8% O2) or normoxic (20% O2) conditions. Measurements of IDO and TDO mRNA and protein expression, as well as a number of other kynurenine pathway enzymes, were significantly lower in the explants exposed to hypoxia compared to the controls. Kynurenine output was also significantly reduced in hypoxic conditions in both the first and third trimester explants [97]. These results establish that the placental kynurenine synthetic pathway is downregulated by hypoxic conditions ex vivo. An association has also been found between FGR pregnancies and down regulation of the kynurenine synthetic pathway in third trimester placental tissue. Both gene and protein expressions of IDO and TDO are significantly lower in FGR-affected placentae compared to controls [97]. It is therefore evident that FGR and placental hypoxia are

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associated with downregulation of the kynurenine synthetic pathway. However, the effects of placental hypoxia on the 5-HT pathway are largely unknown. It is suggested that IDO may act as a ‘sink’ for superoxide [97], since IDO is known to utilise the superoxide anion for its activity [98]. A decrease in IDO expression as a result of hypoxia [97] may therefore lead to decreased clearance of superoxide and an inflammatory response, potentially increasing placental 5-HT synthesis. Alternatively, decreased kynurenine synthesis as a result of hypoxia may shift the tryptophan metabolism pathway in favour of 5-HT synthesis. 6. Nutritional factors and their regulation of the 5-HT synthetic pathway Several nutritional factors are proposed to modulate 5-HT synthesis, release and function in peripheral tissues and in the brain. These factors include vitamin D and the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [58]. Deficiencies in these factors during development, combined with genetic elements, are believed to result in dysfunctional 5-HT synthesis and function, and may be an underlying mechanism that contributes to the development of neurodevelopmental disorders [58]. Sufficient EPA levels in the brain appear to be important for regulation of 5-HT release in the pre-synaptic neuron, and DHA is thought to mediate regulation of 5-HT receptor function [58]. Vitamin D deficiency is thought to repress TPH1 transcription in peripheral tissues and activate TPH2 transcription in the brain. This follows from the identification of two different vitamin D response elements (VDREs) in the genes responsible for TPH1 and TPH2 transcription [54], and evidence that the VDRE sequence can determine the activation or repression of gene transcription by vitamin D [99]. As such, vitamin D supplementation may play a role in optimising low placental 5-HT levels during pregnancy, and this is an area for future investigation. 7. Conclusion FGR, a condition often caused by placental insufficiency, is associated with a number of serious complications, including neurodevelopmental disorders in offspring. Since 5-HT is synthesised in the placenta during pregnancy and appears to linked to brain development, understanding the factors that disturb 5-HT synthesis may be of clinical significance. Also unclear is whether disruption of the placental 5-HT pathway contributes to the pathogenesis of FGR. Preliminary animal and in vitro studies have shown promising associations between placental insults involved in FGR and disrupted tryptophan metabolism. As such, investigating the association between FGR and a disrupted placental 5-HT synthetic pathway in humans comprises an important direction for future research. Acknowledgments We wish to acknowledge the funding support from The Australian Institute for Musculo-Skeletal Science (AIMSS), The University of Melbourne, Victoria, Australia. References [1] L.M.M. Nardozza, et al., Fetal growth restriction: current knowledge, Arch. Gynecol. Obstet. (2017) 1e17. [2] U. Krishna, S. Bhalerao, Placental insufficiency and fetal growth restriction, J. Obstet. Gynaecol. India 61 (5) (2011) 505e511. [3] C.M. Scifres, D.M. Nelson, Intrauterine growth restriction, human placental development and trophoblast cell death, J. Physiol. 587 (14) (2009)

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