Allicin in pregnancy diets modulates steroid metabolism in pregnant sows and placental sulphate metabolism promoting placental angiogenesis and foetal development.

The low-birth-weight of piglets is an important factor affecting pig enterprises. The placenta, as a key organ for material exchange between mother and foetus, directly influences the growth and development of the foetus. Allicin exhibits various biological activities, including anti-inflammatory and antioxidant properties. It may also play a crucial role in enhancing sow reproductive performance and placental angiogenesis. In this study, we used 70 lactating Landrace × Yorkshire binary heterozygous sows to explore the effect of allicin on the reproductive performance of sows and placental development. The sows were randomly assigned into the Allicin group (Allicin), which was fed with a diet containing 0.25% allicin, and the negative control group, which was fed with basal feed. The experimental period lasted for 114 d from the date of mating to the end of farrowing. The results showed that the addition of allicin to the gestation diets increased the number of total born piglets, born alive piglets, and high-birth-weight piglets, reduced peripartum oxidative stress, alleviated dysregulation of glucose-lipid metabolism in sows, and increased the levels of antioxidant markers in the placenta. Differential analysis of metabolites in maternal plasma and placenta samples by non-targeted metabolomics revealed that allicin improved cholesterol metabolism, steroid biosynthesis, and increased plasma progesterone levels in sows. Allicin promoted sulphur metabolism, cysteine and methionine metabolism in placental samples and increased the hydrogen sulphide (H2S) content in the placenta. In addition, Quantitative Real-time PCR, Western blot and immunofluorescence results showed that allicin upregulated the expression of angiogenesis-related genes, VEGF-A, FLK 1 and Ang 1, in the placenta, implying that it promoted placental angiogenesis. These results indicate that supplementing the diet of pregnant sows with allicin reduces oxidative stress, alleviates dysregulation of glucose-lipid metabolism during the periparturient period, and promotes placental angiogenesis and foetal development by increasing plasma progesterone level and placental H2S content.

Maternal Iron Deficiency Alters Trophoblast Differentiation and Placental Development in Rat Pregnancy.

Iron deficiency, which occurs when iron demands chronically exceed intake, is prevalent in pregnant women. Iron deficiency during pregnancy poses major risks for the baby, including fetal growth restriction and long-term health complications. The placenta serves as the interface between a pregnant mother and her baby, and it ensures adequate nutrient provisions for the fetus. Thus, maternal iron deficiency may impact fetal growth and development by altering placental function. We used a rat model of diet-induced iron deficiency to investigate changes in placental growth and development. Pregnant Sprague-Dawley rats were fed either a low-iron or iron-replete diet starting 2 weeks before mating. Compared with controls, both maternal and fetal hemoglobin were reduced in dams fed low-iron diets. Iron deficiency decreased fetal liver and body weight, but not brain, heart, or kidney weight. Placental weight was increased in iron deficiency, due primarily to expansion of the placental junctional zone. The stimulatory effect of iron deficiency on junctional zone development was recapitulated in vitro, as exposure of rat trophoblast stem cells to the iron chelator deferoxamine increased differentiation toward junctional zone trophoblast subtypes. Gene expression analysis revealed 464 transcripts changed at least 1.5-fold (P < 0.05) in placentas from iron-deficient dams, including altered expression of genes associated with oxygen transport and lipoprotein metabolism. Expression of genes associated with iron homeostasis was unchanged despite differences in levels of their encoded proteins. Our findings reveal robust changes in placentation during maternal iron deficiency, which could contribute to the increased risk of fetal distress in these pregnancies.

Maternal high-fat diet during pregnancy with concurrent phthalate exposure leads to abnormal placentation.

Di(2-ethylhexyl) phthalate (DEHP) is a synthetic chemical commonly used for its plasticizing capabilities. Because of the extensive production and use of DEHP, humans are exposed to this chemical daily. Diet is a significant exposure pathway and fatty food contain the highest level of phthalates. The impact on pregnancy following DEHP exposure and the associated interaction of high fat (HF) diet remains unknown. Here we report that exposure of pregnant mice to an environmentally relevant level of DEHP did not affect pregnancy. In contrast, mice fed a HF diet during gestation and exposed to the same level of DEHP display marked impairment in placental development, resulting in poor pregnancy outcomes. Our study further reveals that DEHP exposure combined with a HF diet interfere with the signaling pathway controlled by nuclear receptor PPARγ to adversely affect differentiation of trophoblast cells, leading to compromised vascularization and glucose transport in the placenta. Collectively, these findings demonstrate that maternal diet during pregnancy is a critical factor that determines whether exposure to an environmental toxicant results in impaired placental and fetal development, causing intrauterine growth restriction, fetal morbidity, and mortality.

Novel Technologies for Target Delivery of Therapeutics to the Placenta during Pregnancy: A Review.

Uterine spiral artery remodeling is essential for placental perfusion and fetal growth and, when impaired, results in placental ischemia and pregnancy complications, e.g., fetal growth restriction, preeclampsia, premature birth. Despite the high incidence of adverse pregnancies, current treatment options are limited. Accordingly, research has shifted to the development of gene therapy technologies that provide targeted delivery of "payloads" to the placenta while limiting maternal and fetal exposure. This review describes the current strategies, including placental targeting peptide-bound liposomes, nanoparticle or adenovirus constructs decorated with specific peptide sequences and placental gene promoters delivered via maternal IV injection, directly into the placenta or the uterine artery, as well as noninvasive site-selective targeting of regulating genes conjugated with microbubbles via contrast-enhanced ultrasound. The review also provides a perspective on the effectiveness of these technologies in various animal models and their practicability and potential use for targeted placental delivery of therapeutics and genes in adverse human pregnancies affected by placental dysfunction.

Perfluoroalkyl Substance Exposure and the BDNF Pathway in the Placental Trophoblast.

Background: Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants that have become globally ubiquitous in humans and the environment. In utero PFAS exposure is associated with neurodevelopmental effects; however, the mechanism is poorly understood. Brain-derived neurotrophic factor (BDNF) signaling is critical to fetal neurodevelopment during pregnancy and maintains important regulatory roles later in life. This study aims to characterize placental BDNF signaling and investigate whether PFAS exposure disrupts the signaling pathway in placental trophoblast cells. Methods: The expression and localization of BDNF receptors-p75NTR and TrkB-in first trimester and term human placentas and trophoblast cells were investigated by immunofluorescence staining. To assess the effects of PFAS exposure on the BDNF pathway, BeWo cells were treated with PFAS mixtures that mimicked blood levels in a highly exposed population and major PFAS compounds in the mixture at 0.01, 0.1, 1, and 10 µM concentrations. Changes in pro-BDNF levels and phosphorylation of TrkB receptors were examined by Western blot. Results: In first trimester human placentas, TrkB and p75NTR receptors were primarily localized to syncytiotrophoblast and cytotrophoblast cells. At term, TrkB and p75NTR receptors were primarily observed in the placental villous stroma. TrkB receptor staining in trophoblasts was reduced at term, while p75NTR receptor staining was negative. TrkB receptors were confined to the nuclear and perinuclear spaces, and phosphorylation occurred at the Tyr816 residue in BeWo cells. Exposure to PFOS, PFOA, PFBS, and the six-PFAS mixture did not significantly affect BDNF levels or activation (phosphorylation) of TrkB. Treating cells with 1 µM and 10 µM of PFNA resulted in increased TrkB phosphorylation compared to unexposed controls, but BDNF levels were unchanged. Conclusions: BDNF receptors are present in different regions of human placental villi, indicating diverse functions of BDNF signaling in placental development. Our findings suggest that the BDNF pathway in placental trophoblast cells is not disrupted by exposures to PFOS, PFOA, PFBS, and a PFAS mixture, but may be affected by PFNA exposures. Further investigation is needed on how PFAS affects other critical signaling pathways during fetal neurodevelopment.

Syncytialization alters the extracellular matrix and barrier function of placental trophoblasts.

The human placenta is of vital importance for proper nutrient and waste exchange, immune regulation, and overall fetal health and growth. Specifically, the extracellular matrix (ECM) of placental syncytiotrophoblasts, which extends outward from the placental chorionic villi into maternal blood, acts on a molecular level to regulate and maintain this barrier. Importantly, placental barrier dysfunction has been linked to diseases of pregnancy such as preeclampsia and intrauterine growth restriction. To help facilitate our understanding of the interface and develop therapeutics to repair or prevent dysfunction of the placental barrier, in vitro models of the placental ECM would be of great value. In this study, we aimed to characterize the ECM of an in vitro model of the placental barrier using syncytialized BeWo choriocarcinoma cells. Syncytialization caused a marked change in syndecans, integral proteoglycans of the ECM, which matched observations of in vivo placental ECM. Syndecan-1 expression increased greatly and predominated the other variants. Barrier function of the ECM, as measured by electric cell-substrate impedance sensing (ECIS), increased significantly during and after syncytialization, whereas the ability of THP-1 monocytes to adhere to syncytialized BeWos was greatly reduced compared with nonsyncytialized controls. Furthermore, ECIS measurements indicated that ECM degradation with matrix metalloproteinase-9 (MMP-9), but not heparanase, decreased barrier function. This decrease in ECIS-measured barrier function was not associated with any changes in THP-1 adherence to syncytialized BeWos treated with heparanase or MMP-9. Thus, syncytialization of BeWos provides a physiologically accurate placental ECM with a barrier function matching that seen in vivo.

Maternal Exposure to Oxidized Soybean Oil Impairs Placental Development by Modulating Nutrient Transporters in a Rat Model.

INTRODUCTION: As an exogenous food contaminant, dietary oxidized lipid impairs growth and development, and triggers chronic diseases in humans or animals. This study explores the effects of soybean oil with different oxidative degree on the placental injury of gestational rats. METHODS AND RESULTS: Thirty-two female adult rats are randomly assigned to four groups. The control group is fed the purified diet with fresh soybean oil (FSO), and the treatment groups are fed purified diets with lipid content replaced by oxidized soybean oil (OSO) at 200, 400, and 800 mEqO2 kg-1 from conception until delivery. On day 20 of gestation, OSO decreased placental and embryonic weights as the oxidative degree increased linearly and quadratically. The expression of Bax showed a linear increase, and Bcl-2 decreased as the oxidative degree increased. The expression of Fosl1 and Esx1 is linearly and quadratically decreased in OSO-treated groups than FSO group. OSO decreased the level of IL-10 but increased expression of IL-1ß in placenta and plasma. OSO remarkably upregulates levels of Fatp1 and Glut1 and decreases expression of Snat2 and Glut3. CONCLUSION: OSO aggravates placental injury by modulating nutrient transporters and apoptosis-related genes, impedes placental growth and development, and ultimately leads to the decrease of fetal weight.

FURIN and placental syncytialisation: a cautionary tale.

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.

Perigestational alcohol consumption induces altered early placentation and organogenic embryo growth restriction by disruption of trophoblast angiogenic factors.

RESEARCH QUESTION: Maternal alcohol consumption produces fetal retardation and malformations, probably associated with placental defects. Does perigestational alcohol consumption up to organogenesis lead to abnormal placentation and embryo growth restriction by disrupting the vascular endothelial growth factor (VEGF) system in embryo-placental development? DESIGN: Female mice were treated with 10% ethanol in drinking water before and up to day 10 of gestation. Control mice received ethanol-free water. After treatment, the trophoblastic tissue, embryo growth and the angiogenic VEGF pathway were analysed. RESULTS: Female mice who had received treatment had resorbed and delayed implantation sites with poor ectoplacental cone development. Reduced trophoblastic area tissue from female mice who had received treatment had abnormal junctional zone and diminished labyrinthine vascularization. After treatment, the labyrinth had increased chorionic trophoblast proliferation, hypoxia inducible factor-1α immunoexpression but reduced apoptosis. The embryo growth was reduced concomitantly with low VEGF immunostaining but high endothelial nitric oxide synthase (eNOS) expression. In junctional and labyrinth of treated female mice, gene and protein immunoexpression of VEGF was reduced and the protein expression of FLT-1 increased compared with controls. Increased activation of kinase insert domain receptor receptor (phosphorylated KDR) and expression of eNOS were observed in placenta of treated female mice. Immunoexpression of metalloproteinase-9, however, was reduced in junctional zone but increased in labyrinth, compared with controls. CONCLUSIONS: These data reveal inadequate expression of VEGF/receptors and angiogenic eNOS and metalloproteinase factors related to abnormal early placentation after perigestational alcohol ingestion, providing insight into aetiological factors underlying early placentopathy associated with intrauterine growth restriction caused by maternal alcohol consumption.

Endocannabinoid signaling impairs syncytialization: Using flow cytometry to evaluate forskolin-induced cell fusion.

Cytotrophoblast cells fuse to form the syncytiotrophoblast, the main structure responsible for the placenta's specialized functions. This complex process denominated syncytialization is fundamental for a correct pregnancy outcome. We observed that the endocannabinoid anandamide disrupts syncytialization employing traditional techniques and flow cytometry in BeWo cell line.

Gene expression in rat placenta after exposure to di(2-ethylhexyl) phthalate.

The organic compound di(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in many products. Exposure to DEHP has been reported to lead to adverse pregnancy outcomes by suppressing placenta growth and development. The aim of this study was to determine the gene expression profiles of rat placenta exposed to (DEHP) and identify genes crucial for the DEHP response. Three groups of Wistar rats were administered an intragastric dose of 1,000 mg/kg DEHP, 500 mg/kg DEHP, or corn oil, RNA was isolated from placenta tissue, and hybridization was performed. Gene expression profiles were analyzed by identifying functional enrichment, differentially expressed genes (DEGs), protein-protein interaction (PPI) networks and modules, and transcription factor (TF)-miRNA-target regulatory networks. We obtained 2,032 DEGs, including cytochrome P450, family 2, subfamily R, polypeptide 1 (CYP2R1), sterol O-acyltransferase 2 (SOAT2), and 24-dehydrocholesterol reductase (DHCR24) from the steroid biosynthesis pathway and somatostatin receptor 4 (SSTR4) and somatostatin receptor 2 (SSTR2) in the neuroactive ligand-receptor interaction pathway. The PPI network included 476 nodes, 2,682 interaction pairs, and three sub-network modules. Moreover, eight miRNAs, three TFs, and 176 regulatory pairs were obtained from the TF-miRNA-target regulatory network. CYP2R1, SOAT2, DHCR24, SSTR4, and SSTR2 may affect DEHP influence on rat placenta development.

Periconceptional exposure to lopinavir, but not darunavir, impairs decidualization: a potential mechanism leading to poor birth outcomes in HIV-positive pregnancies.

STUDY QUESTION: Does HIV protease inhibitor (PI)-based combination antiretroviral therapy (cART) initiated at periconception affect key events in early pregnancy, i.e. decidualization and spiral artery remodeling? SUMMARY ANSWER: Two PIs, lopinavir and darunavir, currently offered as cART options in HIV-positive pregnancies were evaluated, and we found that lopinavir-based cART, but not darunavir-based cART, impaired uterine decidualization and spiral artery remodeling in both human ex vivo and mouse in vivo experimental models. WHAT IS KNOWN ALREADY: Early initiation of cART is recommended for pregnant women living with HIV. However, poor birth outcomes are frequently observed in HIV-positive pregnancies exposed to PI-based cART, especially when it is initiated prior to conception. The correlation between early initiation of PI-cART and adverse birth outcomes is poorly understood, due to lack of data on the specific effects of PI-cART on the early stages of pregnancy involving uterine decidualization and spiral artery remodeling. STUDY DESIGN, SIZE, DURATION: Lopinavir and darunavir were evaluated in clinically relevant combinations using an ex vivo human first-trimester placenta-decidua explant model, an in vitro human primary decidual cell culture system, and an in vivo mouse pregnancy model. The first-trimester (gestational age, 6-8 weeks) human placenta-decidua tissue was obtained from 11 to 15 healthy women undergoing elective termination of pregnancy. C57Bl/6 female mice (four/treatment group) were administered either lopinavir-cART, darunavir-cART or water by oral gavage once daily starting on the day of plug detection until sacrifice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human: Spiral artery remodeling was assessed by immunohistochemical analysis of first-trimester placenta-decidua explant co-culture system. Trophoblast migration was measured using a placental explant culture. A primary decidual cell culture was used to evaluate the viability of immune cell populations by flow cytometry. Soluble factors, including biomarkers of decidualization and angiogenesis, were quantified by ELISA and Luminex assay using decidua-conditioned media. Mouse: In the mouse pregnancy model, gestational day 6.5 or 9.5 implantation sites were used to assess decidualization, spiral artery remodeling and uterine natural killer (uNK) cell numbers by immunohistochemistry. Transcription factor STAT3 was assayed by immunohistochemistry in both human decidua and mouse implantation sites. MAIN RESULTS AND THE ROLE OF CHANCE: Lopinavir-cART, but not darunavir-cART, impaired uterine decidualization and spiral artery remodeling in both experimental models. Lopinavir-cART treatment was also associated with selective depletion of uNK cells, reduced trophoblast migration and defective placentation. The lopinavir-associated decidualization defects were attributed to a decrease in expression of transcription factor STAT3, known to regulate decidualization. Our results suggest that periconceptional initiation of lopinavir-cART, but not darunavir-cART, causes defective maturation of the uterine endometrium, leading to impairments in spiral artery remodeling and placentation, thus contributing to the poor birth outcomes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The human first-trimester placenta/decidua samples could only be obtained from healthy females undergoing elective termination of pregnancy. As biopsy is the only way to obtain first-trimester decidua from pregnant women living with HIV on PI-cART, ethics approval and participant consent are difficult to obtain. Furthermore, our animal model is limited to the study of cART and does not include HIV. HIV infection is also associated with immune dysregulation, inflammation, alterations in angiogenic factors and complement activation, all of which could influence decidual and placental vascular remodeling and modify any cART effects. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide mechanistic insight with direct clinical implications, rationalizing why the highest adverse birth outcomes are reported in HIV-positive pregnancies exposed to lopinavir-cART from conception. We demonstrate that dysregulation of decidualization is the mechanism through which lopinavir-cART, but not darunavir-cART, use in early pregnancy leads to poor birth outcomes. Although lopinavir is no longer a first-line regimen in pregnancy, it remains an alternate regimen and is often the only PI available in low resource settings. Our results highlight the need for reconsidering current guidelines recommending lopinavir use in pregnancy and indicate that lopinavir should be avoided especially in the first trimester, whereas darunavir is safe to use and should be the preferred PI in pregnancy.Further, in current times of the COVID-19 pandemic, lopinavir is among the top drug candidates which are being repurposed for inclusion in clinical trials world-over, to assess their therapeutic potential against the dangerous respiratory disease. Current trials are also testing the efficacy of lopinavir given prophylactically to protect health care workers and people with potential exposures. Given the current extraordinary numbers, these might include women with early pregnancies, who may or may not be cognizant of their gestational status. This is a matter of concern as it could mean that women with early pregnancies might be exposed to this drug, which can cause decidualization defects. Our findings provide evidence of safety concerns surrounding lopinavir use in pregnancy, that women of reproductive age considering participation in such trials should be made aware of, so they can make a fully informed decision. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Canadian Institutes of Health Research (CIHR) (PJT-148684 and MOP-130398 to L.S.). C.D. received support from CIHR Foundation (FDN143262 to Stephen Lye). S.K. received a TGHRI postdoctoral fellowship. The authors declare that there are no conflicts of interest. L.S. reports personal fees from ViiV Healthcare for participation in a Women and Transgender Think Tank.

Association of polybrominated diphenyl ether (PBDE) levels with biomarkers of placental development and disease during mid-gestation.

BACKGROUND: Polybrominated diphenyl ether (PBDE) exposures have been associated with adverse pregnancy outcomes. A hypothesized mechanism is via alterations in placental development and function. However, we lack biomarkers that can be used as early indicators of maternal/fetal response to PBDE exposures and/or perturbations in placental development or function. METHODS: To evaluate the relationship between PBDE levels and placental biomarkers during mid-gestation of human pregnancy (n = 62), we immunolocalized three molecules that play key roles in cytotrophoblast (CTB) differentiation and interstitial/endovascular uterine invasion-integrin alpha-1 (ITGA1), vascular endothelial-cadherin (CDH5), and metalloproteinase-1 (MMP1)-and assessed three morphological parameters as potential indicators of pathological alterations using H&E-stained tissues-leukocyte infiltration, fibrinoid deposition, and CTB endovascular invasion. We evaluated associations between placental PBDE levels and of biomarkers of placental development and disease using censored Kendall's tau correlation and linear regression methods. RESULTS: PBDEs were detected in all placental samples. We observed substantial variation in antigen expression and morphological endpoints across placental regions. We observed an association between PBDE concentrations and immunoreactivity of endovascular CTB staining with anti-ITGA1 (inverse) or interstitial CTBs staining with anti-CDH5 (positive). CONCLUSIONS: We found several molecular markers that may be sensitive placental indicators of PBDE exposure. Further, this indicates that placental biomarkers of development and disease could be useful barometers of exposure to PBDEs, a paradigm that could be extended to other environmental chemicals and placental stage-specific antigens.

Vascular Endothelial Growth Factor-121 Administration Mitigates Halogen Inhalation-Induced Pulmonary Injury and Fetal Growth Restriction in Pregnant Mice.

Background Circulating levels of sFLT-1 (soluble fms-like tyrosine kinase 1), the extracellular domain of vascular endothelial growth factor (VEGF) receptor 1, and its ratio to levels of placental growth factor are markers of the occurrence and severity of preeclampsia. Methods and Results C57BL/6 pregnant mice on embryonic day 14.5 (E14.5), male, and non-pregnant female mice were exposed to air or to Br2 at 600 ppm for 30 minutes and were treated with vehicle or with VEGF-121 (100 µg/kg, subcutaneously) daily, starting 48 hours post-exposure. Plasma, bronchoalveolar lavage fluid, lungs, fetuses, and placentas were collected 120 hours post-exposure. In Br2-exposed pregnant mice, there was a time-dependent and significant increase in plasma levels of sFLT-1 which correlated with increases in mouse lung wet/dry weights and bronchoalveolar lavage fluid protein content. Supplementation of exogenous VEGF-121 improved survival and weight gain, reduced lung wet/dry weights, decreased bronchoalveolar lavage fluid protein levels, enhanced placental development, and improved fetal growth in pregnant mice exposed to Br2. Exogenous VEGF-121 administration had no effect in non-pregnant mice. Conclusions These results implicate inhibition of VEGF signaling driven by sFLT-1 overexpression as a mechanism of pregnancy-specific injury leading to lung edema, maternal mortality, and fetal growth restriction after bromine gas exposure.

The impact of tobacco chemicals and nicotine on placental development.

Despite decades of messages warning about the dangers of tobacco use in pregnancy, 10% to 15% of pregnant women continue to smoke. Furthermore, an increased popularity of electronic nicotine delivery systems (ENDS) over the past decade in women of childbearing age raises parallel concerns regarding the effects of vaporized nicotine use in pregnancy. While research using animal models which mimic tobacco smoke and nicotine exposure in pregnancy have largely replicated findings in humans, few studies focus directly on the effects of these exposures on the placenta. Because the placenta is a fetal derived tissue, and nicotine and other components of tobacco smoke are either processed by or transported directly through the placenta, such studies help us understand the risks of these exposures on the developing fetus. In this review, we summarize research on the placenta and placental-derived cells examining either tobacco smoke or nicotine exposure, including both histologic and subcellular (ie, epigenetic and molecular) modifications. Collectively, these studies reveal that tobacco and nicotine exposure are accompanied by some common and several unique molecular and epigenomic placental modifications. Consideration of the nature and sequelae of these molecular mediators of risk may help to better inform the public and more effectively curtail modifiable behavior.

Nicotine Suppresses the Invasiveness of Human Trophoblasts by Downregulation of CXCL12 Expression through the Alpha-7 Subunit of the Nicotinic Acetylcholine Receptor.

Smoke exposure during pregnancy has detrimental effects upon numerous fetal and neonatal outcomes. Nicotine (the main component of tobacco) has been suggested to affect placental development. During placental development, efficient invasion by trophoblasts is required for establishment of the fetus-maternal circulation. In this study we explored the regulation of trophoblast invasion by nicotine. An immortalized first trimester extravillous trophoblast cell line (HTR-8/SVneo cells) was used for all the experiments, which were treated by nicotine, methyllycaconitine, and C-X-C motif chemokine ligand 12 (CXCL12). Total RNA and protein were used to study the expressions of nicotinic acetylcholine receptors (nAChRs), and transwell assay was used to study invasiveness. Changes of RNA expression due to nicotine treatment were detected by RNA sequence. Level of CXCL12 mRNA was verified by quantitative PCR. We showed that HTR-8/SVneo expressed subunits α2-4, α7, α9, ß1, and ß2 of nAChRs. Nicotine downregulated CXCL12 expression and inhibited trophoblast invasion. Methyllycaconitine, as an antagonist of the α7 homopolymer, blocked the inhibitory effect of nicotine. CXCL12 could rescue the nicotine-induced inhibitory effect on invasion of HTR-8/SVneo cells. These results suggest that the α7 subunit of the nAChR has important roles in modulating trophoblast invasion through CXCL12.

Maternal iron nutriture modulates placental development in a rat model of fetal alcohol spectrum disorder.

Prenatal alcohol exposure (PAE) causes developmental abnormalities known as fetal alcohol spectrum disorder (FASD). Maternal iron status modulates the severity of these defects in the offspring. Because the placenta is central in supporting fetal development, we investigated whether maternal iron status similarly modulates alcohol's effects in the placenta. We hypothesized that PAE causes placental insufficiency by decreasing placental weight and efficiency, and we hypothesized that these are worsened by maternal iron deficiency (ID) and alleviated by dietary iron fortification (IF). We also determined whether altered placental iron flux and inflammatory balance contribute to placental insufficiency. Pregnant Long-Evans rats consumed an iron-deficient (ID; 2-6 ppm), iron-sufficient (IS; 100 ppm), or iron-fortified (IF; 500 ppm) diet. Alcohol (5 g/kg body weight) or isocaloric maltodextrin (MD) was gavaged daily from gestational day (GD) 13.5-19.5. Placental outcomes were evaluated on GD20.5. PAE reduced fetal weight (p < 0.0001), placental weight (p = 0.0324), and placental efficiency (p = 0.0043). PAE downregulated placental transferrin receptor (p = 0.0032); it also altered placental Il1b and Tnf expression and the Il6:Il10 ratio (p = 0.0337, 0.0300, and 0.0034, respectively) to generate a response favoring inflammation. ID-PAE further reduced fetal growth and placental efficiency and induced a heightened pro-inflammatory placental profile. IF did not rescue the alcohol-reduced fetal weight, but it normalized placental efficiency and decreased placental inflammation. These placental cytokines correlated with fetal and placental growth, and explained 45% of the variability in fetal weight and 20% of the variability in placental efficiency. In summary, alcohol induces placental insufficiency and is associated with a pro-inflammatory cytokine profile exacerbated by maternal ID and mitigated by maternal IF. Because the placenta is closely linked to intrauterine growth, the placental insufficiency reported here may correlate with the lower birth weights in a subgroup of individuals who experienced PAE.

Review: Understanding the role of androgens and placental AR variants: Insight into steroid-dependent fetal-placental growth and development.

The role of steroids throughout pregnancy and their effect on placental physiology is well established, especially for estrogens, progestogens, and glucocorticoids; however, the role of androgens - particularly within the context of placental physiology - remains largely unexplored. Androgens are often defined as the male sex-steroids and are fundamental for the defeminisation and masculinisation of male fetuses. Therefore, the placenta may adapt to these steroids in a sex-specific manner, with males being more receptive to changes in these steroids concentrations, when compared with females; however, their involvement in female intrauterine development has been investigated in several studies and may suggest females have a level of responsiveness to these steroids. While the former may be true, studies have reported sex-specific differences in the expression and activity of factors involved in androgen biosynthesis and bioavailability, with males consistently demonstrating greater degrees of altered protein and gene expression when compared with females. Understanding the placental androgen axis is essential as many pregnancy comorbidities are associated with elevated concentrations of androgens and perturbed intrauterine development or growth. Indeed, it appears that specific pathophysiologies of pregnancy can modulate the activity of key factors involved in the placental androgen axis and this may contribute to the etiology of sex-specific developmental outcomes from certain pregnancy complications. This review will provide insight into what is currently known regarding androgen signalling and the human placenta, and how this complex system may regulate sex-specific growth and developmental outcomes in normal and adverse pregnancies.

Maternal circulating miRNAs that predict infant FASD outcomes influence placental maturation.

Prenatal alcohol exposure (PAE), like other pregnancy complications, can result in placental insufficiency and fetal growth restriction, although the linking causal mechanisms are unclear. We previously identified 11 gestationally elevated maternal circulating miRNAs (HEamiRNAs) that predicted infant growth deficits following PAE. Here, we investigated whether these HEamiRNAs contribute to the pathology of PAE, by inhibiting trophoblast epithelial-mesenchymal transition (EMT), a pathway critical for placental development. We now report for the first time that PAE inhibits expression of placental pro-EMT pathway members in both rodents and primates, and that HEamiRNAs collectively, but not individually, mediate placental EMT inhibition. HEamiRNAs collectively, but not individually, also inhibited cell proliferation and the EMT pathway in cultured trophoblasts, while inducing cell stress, and following trophoblast syncytialization, aberrant endocrine maturation. Moreover, a single intravascular administration of the pooled murine-expressed HEamiRNAs, to pregnant mice, decreased placental and fetal growth and inhibited the expression of pro-EMT transcripts in the placenta. Our data suggest that HEamiRNAs collectively interfere with placental development, contributing to the pathology of PAE, and perhaps also, to other causes of fetal growth restriction.

Hyperandrogenism and insulin resistance induce gravid uterine defects in association with mitochondrial dysfunction and aberrant reactive oxygen species production.

Women with polycystic ovary syndrome (PCOS) are at increased risk of miscarriage, which often accompanies the hyperandrogenism and insulin resistance seen in these patients. However, neither the combinatorial interaction between these two PCOS-related etiological factors nor the mechanisms of their actions in the uterus during pregnancy are well understood. We hypothesized that hyperandrogensim and insulin resistance exert a causative role in miscarriage by inducing defects in uterine function that are accompanied by mitochondrial-mediated oxidative stress, inflammation, and perturbed gene expression. Here, we tested this hypothesis by studying the metabolic, endocrine, and uterine abnormalities in pregnant rats after exposure to daily injection of 5α-dihydrotestosterone (DHT; 1.66 mg·kg body wt-1·day-1) and/or insulin (6.0 IU/day) from gestational day 7.5 to 13.5. We showed that whereas DHT-exposed and insulin-exposed pregnant rats presented impaired insulin sensitivity, DHT + insulin-exposed pregnant rats exhibited hyperandrogenism and peripheral insulin resistance, which mirrors pregnant PCOS patients. Compared with controls, hyperandrogenism and insulin resistance in the dam were associated with alterations in uterine morphology and aberrant expression of genes responsible for decidualization (Prl8a2, Fxyd2, and Mt1g), placentation (Fcgr3 and Tpbpa), angiogenesis (Flt1, Angpt1, Angpt2, Ho1, Ccl2, Ccl5, Cxcl9, and Cxcl10) and insulin signaling (Akt, Gsk3, and Gluts). Moreover, we observed changes in uterine mitochondrial function and homeostasis (i.e., mitochondrial DNA copy number and the expression of genes responsible for mitochondrial fusion, fission, biogenesis, and mitophagy) and suppression of both oxidative and antioxidative defenses (i.e., reactive oxygen species, Nrf2 signaling, and interactive networks of antioxidative stress responses) in response to the hyperandrogenism and insulin resistance. These findings demonstrate that hyperandrogenism and insulin resistance induce mitochondria-mediated damage and a resulting imbalance between oxidative and antioxidative stress responses in the gravid uterus.