Prenatal inflammation as a link between placental expression signature of tryptophan metabolism and preterm birth.

Spontaneous preterm birth is a serious medical condition responsible for substantial perinatal morbidity and mortality. Its phenotypic characteristics, preterm labor with intact membranes (PTL) and preterm premature rupture of the membranes (PPROM), are associated with significantly increased risks of neurological and behavioral alterations in childhood and later life. Recognizing the inflammatory milieu associated with PTL and PPROM, here, we examined expression signatures of placental tryptophan metabolism, an important pathway in prenatal brain development and immunotolerance. The study was performed in a well-characterized clinical cohort of healthy term pregnancies (n = 39) and 167 preterm deliveries (PTL, n = 38 and PPROM, n = 129). Within the preterm group, we then investigated potential mechanistic links between differential placental tryptophan pathway expression, preterm birth and both intra-amniotic markers (such as amniotic fluid interleukin-6) and maternal inflammatory markers (such as maternal serum C-reactive protein and white blood cell count). We show that preterm birth is associated with significant changes in placental tryptophan metabolism. Multifactorial analysis revealed similarities in expression patterns associated with multiple phenotypes of preterm delivery. Subsequent correlation computations and mediation analyses identified links between intra-amniotic and maternal inflammatory markers and placental serotonin and kynurenine pathways of tryptophan catabolism. Collectively, the findings suggest that a hostile inflammatory environment associated with preterm delivery underlies the mechanisms affecting placental endocrine/transport functions and may contribute to disruption of developmental programming of the fetal brain.

Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods.

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Characterization of a human placental clearance system to regulate serotonin levels in the fetoplacental unit.

BACKGROUND: Serotonin (5-HT) is a biogenic monoamine with diverse functions in multiple human organs and tissues. During pregnancy, tightly regulated levels of 5-HT in the fetoplacental unit are critical for proper placental functions, fetal development, and programming. Despite being a non-neuronal organ, the placenta expresses a suite of homeostatic proteins, membrane transporters and metabolizing enzymes, to regulate monoamine levels. We hypothesized that placental 5-HT clearance is important for maintaining 5-HT levels in the fetoplacental unit. We therefore investigated placental 5-HT uptake from the umbilical circulation at physiological and supraphysiological levels as well as placental metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA efflux from trophoblast cells. METHODS: We employed a systematic approach using advanced organ-, tissue-, and cellular-level models of the human placenta to investigate the transport and metabolism of 5-HT in the fetoplacental unit. Human placentas from uncomplicated term pregnancies were used for perfusion studies, culturing explants, and isolating primary trophoblast cells. RESULTS: Using the dually perfused placenta, we observed a high and concentration-dependent placental extraction of 5-HT from the fetal circulation. Subsequently, within the placenta, 5-HT was metabolized to 5-hydroxyindoleacetic acid (5-HIAA), which was then unidirectionally excreted to the maternal circulation. In the explant cultures and primary trophoblast cells, we show concentration- and inhibitor-dependent 5-HT uptake and metabolism and subsequent 5-HIAA release into the media. Droplet digital PCR revealed that the dominant gene in all models was MAO-A, supporting the crucial role of 5-HT metabolism in placental 5-HT clearance. CONCLUSIONS: Taken together, we present transcriptional and functional evidence that the human placenta has an efficient 5-HT clearance system involving (1) removal of 5-HT from the fetal circulation by OCT3, (2) metabolism to 5-HIAA by MAO-A, and (3) selective 5-HIAA excretion to the maternal circulation via the MRP2 transporter. This synchronized mechanism is critical for regulating 5-HT in the fetoplacental unit; however, it can be compromised by external insults such as antidepressant drugs.

Perfused rat term placenta as a preclinical model to investigate placental dopamine and norepinephrine transport.

The placenta represents a non-neuronal organ capable of transporting and metabolizing monoamines. Since these bioactive molecules participate in numerous processes essential for placental and fetal physiology, any imbalance in their levels during pregnancy may affect brain development, projecting a higher risk of behavioral disorders in childhood or adulthood. Notably, the monoamine system in the placenta is a target of various psychoactive drugs and can be disrupted in several pregnancy pathologies. As research in pregnant women poses significant ethical restrictions, animal models are widely employed to study monoamine homeostasis as a mechanism involved in fetal programming. However, detailed knowledge of monoamine transport in the rat placenta is still lacking. Moreover, relatability to the human placental monoamine system is not examined. The present study provides insights into the transplacental monoamine dynamics between maternal and fetal circulation. We show that norepinephrine maternal-to-fetal transport is <4% due to high metabolism within the trophoblast. In contrast, dopamine maternal-to-fetal transport exceeds 25%, likely through passive transport across the membrane. In addition, we show high clearance of norepinephrine and dopamine from the fetal circulation mediated by the organic cation transporter 3 (OCT3). Altogether, we present transcriptional and functional evidence that the in situ rat placenta perfusion represents a suitable model for (patho)physiological investigation of dopamine and norepinephrine homeostasis in the fetoplacental unit. With the rapid advancements in drug discovery and environmental toxicity, the use of rat placenta as a preclinical model could facilitate screening of possible xenobiotic effects on monoamine homeostasis in the placenta.

Profiling of Tryptophan Metabolic Pathways in the Rat Fetoplacental Unit During Gestation.

Placental homeostasis of tryptophan is essential for fetal development and programming. The two main metabolic pathways (serotonin and kynurenine) produce bioactive metabolites with immunosuppressive, neurotoxic, or neuroprotective properties and their concentrations in the fetoplacental unit must be tightly regulated throughout gestation. Here, we investigated the expression/function of key enzymes/transporters involved in tryptophan pathways during mid-to-late gestation in rat placenta and fetal organs. Quantitative PCR and heatmap analysis revealed the differential expression of several genes involved in serotonin and kynurenine pathways. To identify the flux of substrates through these pathways, Droplet Digital PCR, western blot, and functional analyses were carried out for the rate-limiting enzymes and transporters. Our findings show that placental tryptophan metabolism to serotonin is crucial in mid-gestation, with a subsequent switch to fetal serotonin synthesis. Concurrently, at term, the close interplay between transporters and metabolizing enzymes of both placenta and fetal organs orchestrates serotonin homeostasis and prevents hyper/hypo-serotonemia. On the other hand, the placental production of kynurenine increases during pregnancy, with a low contribution of fetal organs throughout gestation. Any external insult to this tightly regulated harmony of transporters and enzymes within the fetoplacental unit may affect optimal in utero conditions and have a negative impact on fetal programming.

Functional characterization of dopamine and norepinephrine transport across the apical and basal plasma membranes of the human placental syncytiotrophoblast.

The human placenta represents a unique non-neuronal site of monoamine transporter expression, with pathophysiological relevance during the prenatal period. Monoamines (serotonin, dopamine, norepinephrine) are crucial neuromodulators for proper placenta functions and fetal development, including cell proliferation, differentiation, and neuronal migration. Accumulating evidence suggests that even a transient disruption of monoamine balance during gestation may lead to permanent changes in the fetal brain structures and functions, projecting into adulthood. Nonetheless, little is known about the transfer of dopamine and norepinephrine across the placental syncytiotrophoblast. Employing the method of isolated membranes from the human term placenta, here we delineate the transport mechanisms involved in dopamine and norepinephrine passage across the apical microvillous (MVM) and basal membranes. We show that the placental uptake of dopamine and norepinephrine across the mother-facing MVM is mediated via the high-affinity and low-capacity serotonin (SERT/SLC6A4) and norepinephrine (NET/SLC6A2) transporters. In the fetus-facing basal membrane, however, the placental uptake of both monoamines is controlled by the organic cation transporter 3 (OCT3/SLC22A3). Our findings thus provide insights into physiological aspects of dopamine and norepinephrine transport across both the maternal and fetal sides of the placenta. As monoamine transporters represent targets for several neuroactive drugs such as antidepressants, our findings are pharmacologically relevant to ensure the safety of drug use during pregnancy.

Effect of Selected Antidepressants on Placental Homeostasis of Serotonin: Maternal and Fetal Perspectives.

Depression is a prevalent condition affecting up to 20% of pregnant women. Hence, more than 10% are prescribed antidepressant drugs, mainly serotonin reuptake inhibitors (SSRIs) and selective serotonin and noradrenaline reuptake inhibitors (SNRIs). We hypothesize that antidepressants disturb serotonin homeostasis in the fetoplacental unit by inhibiting serotonin transporter (SERT) and organic cation transporter 3 (OCT3) in the maternal- and fetal-facing placental membranes, respectively. Paroxetine, citalopram, fluoxetine, fluvoxamine, sertraline, and venlafaxine were tested in situ (rat term placenta perfusion) and ex vivo (uptake studies in membrane vesicles isolated from healthy human term placenta). All tested antidepressants significantly inhibited SERT- and OCT3-mediated serotonin uptake in a dose-dependent manner. Calculated half-maximal inhibitory concentrations (IC50) were in the range of therapeutic plasma concentrations. Using in vitro and in situ models, we further showed that the placental efflux transporters did not compromise mother-to-fetus transport of antidepressants. Collectively, we suggest that antidepressants have the potential to affect serotonin levels in the placenta or fetus when administered at therapeutic doses. Interestingly, the effect of antidepressants on serotonin homeostasis in rat placenta was sex dependent. As accurate fetal programming requires optimal serotonin levels in the fetoplacental unit throughout gestation, inhibition of SERT-/OCT3-mediated serotonin uptake may help explain the poor outcomes of antidepressant use in pregnancy.

Dynamics of Tryptophan Metabolic Pathways in Human Placenta and Placental-Derived Cells: Effect of Gestation Age and Trophoblast Differentiation.

L-Tryptophan is an essential amino acid and a precursor of several physiologically active metabolites. In the placenta, the serotonin and kynurenine metabolic pathways of tryptophan metabolism have been identified, giving rise to various molecules of neuroactive or immunoprotective properties, such as serotonin, melatonin, kynurenine, kynurenic acid, or quinolinic acid. Current literature suggests that optimal levels of these molecules in the fetoplacental unit are crucial for proper placenta functions, fetal development and programming. Placenta is a unique endocrine organ that, being equipped with a battery of biotransformation enzymes and transporters, precisely orchestrates homeostasis of tryptophan metabolic pathways. However, because pregnancy is a dynamic process and placental/fetal needs are continuously changing throughout gestation, placenta must adapt to these changes and ensure proper communication in the feto-placental unit. Therefore, in this study we investigated alterations of placental tryptophan metabolic pathways throughout gestation. Quantitative polymerase chain reaction (PCR) analysis of 21 selected genes was carried out in first trimester (n = 13) and term (n = 32) placentas. Heatmap analysis with hierarchical clustering revealed differential gene expression of serotonin and kynurenine pathways across gestation. Subsequently, digital droplet PCR, Western blot, and functional analyses of the rate-limiting enzymes suggest preferential serotonin synthesis early in pregnancy with a switch to kynurenine production toward term. Correspondingly, increased function and/or protein expression of serotonin degrading enzyme and transporters at term indicates efficient placental uptake and metabolic degradation of serotonin. Lastly, gene expression analysis in choriocarcinoma-derived cell lines (BeWo, BeWo b30, JEG-3) revealed dissimilar expression patterns and divergent effect of syncytialization compared to primary trophoblast cells isolated from human term placentas; these findings show that the commonly used in vitro placental models are not suitable to study placental handling of tryptophan. Altogether, our data provide the first comprehensive evidence of changes in placental homeostasis of tryptophan and its metabolites as a function of gestational age, which is critical for proper placental function and fetal development.

Serotonin homeostasis in the materno-foetal interface at term: Role of transporters (SERT/SLC6A4 and OCT3/SLC22A3) and monoamine oxidase A (MAO-A) in uptake and degradation of serotonin by human and rat term placenta.

AIM: Serotonin is crucial for proper foetal development, and the placenta has been described as a 'donor' of serotonin for the embryo/foetus. However, in later stages of gestation the foetus produces its own serotonin from maternally-derived tryptophan and placental supply is no longer needed. We propose a novel model of serotonin homeostasis in the term placenta with special focus on the protective role of organic cation transporter 3 (OCT3/SLC22A3). METHODS: Dually perfused rat term placenta was employed to quantify serotonin/tryptophan transport and metabolism. Placental membrane vesicles isolated from human term placenta were used to characterize serotonin transporters on both sides of the syncytiotrophoblast. RESULTS: We obtained the first evidence that serotonin is massively taken up from the foetal circulation by OCT3. This uptake is concentration-dependent and inhibitable by OCT3 blockers of endogenous (glucocorticoids) or exogenous (pharmaceuticals) origin. Population analyses in rat placenta revealed that foetal sex influences placental extraction of serotonin from foetal circulation. Negligible foetal serotonin levels were detected in maternal-to-foetal serotonin/tryptophan transport and metabolic studies. CONCLUSION: We demonstrate that OCT3, localized on the foetus-facing membrane of syncytiotrophoblast, is an essential component of foeto-placental homeostasis of serotonin. Together with serotonin degrading enzyme, monoamine oxidase-A, this offers a protective mechanism against local vasoconstriction effects of serotonin in the placenta. However, this system may be compromised by OCT3 inhibitory molecules, such as glucocorticoids or antidepressants. Our findings open new avenues to explore previously unsuspected/unexplained complications during pregnancy including prenatal glucocorticoid excess and pharmacotherapeutic risks of treating pregnant women with OCT3 inhibitors.