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Elevated glucose levels at the fetal–maternal interface are associated with placental trophoblast dysfunction and increased incidence of pregnancy complications. Trophoblast cells predominantly utilize glucose as an energy source, metabolizing it through glycolysis in the cytoplasm and oxidative respiration in the mitochondria to produce ATP. The TGFβ1/SMAD2 signaling pathway and the transcription factors PPARγ, HIF1α, and AMPK are key regulators of cell metabolism and are known to play critical roles in extravillous trophoblast cell differentiation and function. While HIF1α promotes glycolysis over mitochondrial respiration, PPARγ and AMPK encourage the opposite. However, the interplay between TGFβ1 and these energy-sensing regulators in trophoblast cell glucose metabolism remains unclear. This study aimed to investigate whether and how TGFβ1 regulates energy metabolism in trophoblast cells exposed to normal and high glucose conditions. The trophoblast JEG-3 cells were incubated in normal (5 mM) and high (25 mM) glucose conditions for 24 h in the absence and the presence of TGFβ1. The protein expression levels of phosphor (p)-SMAD2, GLUT1/3, HIF1α, PPARγ, p-AMPK, and specific OXPHOS protein subunits were determined by western blotting, and ATP and lactate production by bioluminescent assay kits. JEG-3 cells exposed to 25 mM glucose decreased ATP production but did not affect lactate production. These changes led to a reduction in the expression levels of GLUT1/3, mitochondrial respiratory chain proteins, and PPARγ, coinciding with an increase in HIF1α expression. Conversely, TGFβ1 treatment at 25 mM glucose reduced HIF1α expression while enhancing the expression levels of GLUT1/3, PPARγ, p-AMPK, and mitochondrial respiratory chain proteins, thereby rejuvenating ATP production. Our findings reveal that high glucose conditions disrupt cellular glucose metabolism in trophoblast cells by perturbing mitochondrial oxidative respiration and decreasing ATP production. Treatment with TGFβ1 appears to counteract this trend, probably by enhancing both glycolytic and mitochondrial metabolism, suggesting a potential regulatory role of TGFβ1 in placental trophoblast cell glucose metabolism. [ABSTRACT FROM AUTHOR]

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

Author(s): Isabelle Sinclair [sup.1] , Myriane St-Pierre [sup.1] , Cathy Vaillancourt [sup.2] , Sonia Gagnon [sup.3] , Kelsey N. Dancause [sup.1] Author Affiliations: (1) grid.38678.32, 0000 0001 2181 0211, Département [...], Introduction Past research shows that stress during pregnancy predicts adverse birth outcomes. These patterns might differ based on immigration status. Our objective was to analyze differences in relationships between perceived stress during pregnancy and birth outcomes by immigration status. Methods We recruited 81 pregnant women in Canada for a prospective longitudinal study of stress during pregnancy and infant development. Participants completed the Perceived Stress Questionnaire at 16-18, 24-26 and 32-34 weeks of pregnancy. Birth records were available for 73 women, including 24 non-immigrants, 18 long-term immigrants ([greater than or equal to] 5 years), and 31 recent immigrants (< 5 years). We used General Linear Models to test relationships between perceived stress and birthweight, birthweight for gestational age Z-scores, and gestational age, and differences based on immigration status. Results Controlling for sociodemographic covariates, we observed interactive relationships between immigration status and perceived stress with birthweight at 16-18 (p = 0.032, partial [eta].sup.2 = 0.11) and 24-26 weeks pregnancy (p = 0.012, partial [eta].sup.2 = 0.15). Results were similar for birthweight for gestational age Z-scores at 16-18 weeks (p = 0.016, partial [eta].sup.2 = 0.13) and 24-26 weeks pregnancy (p = 0.013, partial [eta].sup.2 = 0.14). Perceived stress predicted smaller birthweight measurements among long-term immigrants. No relation was found between perceived stress, immigration status and gestational age. Discussion Risk of adverse health outcomes, including birth outcomes, tends to increase with duration of residence among immigrants. Stress during pregnancy might represent one risk factor for adverse birth outcomes among long-term immigrant women. Promoting psychosocial health screening and care among immigrant women, and assuring continued care with acculturation, might improve both maternal and infant health outcomes.

BackgroundExperimental tools for studying the complex steroidogenic interactions that occur between placenta and fetus during human pregnancy are extremely limited.ObjectivesWe aimed to develop a co-culture model to study steroidogenesis by the human fetoplacental unit and its disruption by exposure to environmental contaminants.MethodsWe cultured BeWo human choriocarcinoma cells, representing the villous cytotrophoblast, and H295R human adrenocortical carcinoma cells, representing the fetal unit, in a carefully adapted co-culture medium. We placed H295R cells in 24-well plates and BeWo cells on transwell inserts with or without pesticide treatment (atrazine or prochloraz) and assessed CYP19 activity and hormonal production after 24 hr of co-culture.ResultsThe co-culture exhibited the steroidogenic profile of the fetoplacental unit, allowing a synergistic production of estradiol and estriol (but not of estrone) of 133.3 ± 11.3 pg/mL and 440.8 ± 44.0 pg/mL, respectively. Atrazine and prochloraz had cell-type specific effects on CYP19 activity and estrogen production in co-culture. Atrazine induced CYP19 activity and estrogen production in H295R cells only, but did not affect overall estrogen production in co-culture, whereas prochloraz inhibited CYP19 activity exclusively in BeWo cells and reduced estrogen production in co-culture by almost 90%. In contrast, prochloraz did not affect estradiol or estrone production in BeWo cells in monoculture. These differential effects underline the relevance of our co-culture approach to model fetoplacental steroidogenesis.ConclusionsThe co-culture of H295R and BeWo cells creates a unique in vitro model to reproduce the steroidogenic cooperation between fetus and placenta during pregnancy and can be used to study the endocrine-disrupting effects of environmental chemicals.

BackgroundExperimental tools for studying the complex steroidogenic interactions that occur between placenta and fetus during human pregnancy are extremely limited.ObjectivesWe aimed to develop a co-culture model to study steroidogenesis by the human fetoplacental unit and its disruption by exposure to environmental contaminants.MethodsWe cultured BeWo human choriocarcinoma cells, representing the villous cytotrophoblast, and H295R human adrenocortical carcinoma cells, representing the fetal unit, in a carefully adapted co-culture medium. We placed H295R cells in 24-well plates and BeWo cells on transwell inserts with or without pesticide treatment (atrazine or prochloraz) and assessed CYP19 activity and hormonal production after 24 hr of co-culture.ResultsThe co-culture exhibited the steroidogenic profile of the fetoplacental unit, allowing a synergistic production of estradiol and estriol (but not of estrone) of 133.3 ± 11.3 pg/mL and 440.8 ± 44.0 pg/mL, respectively. Atrazine and prochloraz had cell-type specific effects on CYP19 activity and estrogen production in co-culture. Atrazine induced CYP19 activity and estrogen production in H295R cells only, but did not affect overall estrogen production in co-culture, whereas prochloraz inhibited CYP19 activity exclusively in BeWo cells and reduced estrogen production in co-culture by almost 90%. In contrast, prochloraz did not affect estradiol or estrone production in BeWo cells in monoculture. These differential effects underline the relevance of our co-culture approach to model fetoplacental steroidogenesis.ConclusionsThe co-culture of H295R and BeWo cells creates a unique in vitro model to reproduce the steroidogenic cooperation between fetus and placenta during pregnancy and can be used to study the endocrine-disrupting effects of environmental chemicals.

The benefits of breastfeeding over formula feed are widely appreciated. However, for many mothers breastfeeding is not possible, highlighting the need for a significant improvement in the contents of formula feed. In this article, the overlooked role of melatonin and the melatonergic pathways in breast milk and in the regulation of wider breast milk components are reviewed. There is a growing appreciation that the benefits of breastfeeding are mediated by its effects in the infant gut, with consequences for the development of the gut-brain axis and the immune system. The melatonergic pathways are intimately associated with highly researched processes in the gut, gut microbiome and gut-brain axis. As the melatonergic pathways are dependent on the levels of serotonin availability as a necessary precursor, decreased melatonin is linked to depression and depression-associated disorders. The association of breastfeeding and the gut-brain axis with a host of medical conditions may be mediated by their regulation of processes that modulate depression susceptibility. The biological underpinnings of depression include increased levels of pro-inflammatory cytokines, oxidative stress, kynurenine pathway activity and dysregulation of the hypothalamic-pituitary adrenal axis, all of which can decrease melatonergic pathway activity. The inclusion of the melatonergic pathways in the biological interactions of breast milk and gut development has significant theoretical and treatment implications, as well as being important to the prevention of a host of infant-, child- and adult-onset medical conditions.

In Figure 3, the H295R and BeWo cell-type labels are inverted between graphs A and B. This does not alter the conclusion that treatment with prochloraz (1 and 3 [micro]M) [...]