Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development.

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

High-throughput mRNA sequencing of human placenta shows sex differences across gestation.

INTRODUCTION: Fetal sex affects fetal and maternal health outcomes in pregnancy, but this connection remains poorly understood. As the placenta is the route of fetomaternal communication and derives from the fetal genome, placental gene expression sex differences may explain these outcomes. OBJECTIVES: We utilized next generation sequencing to study the normal human placenta in both sexes in first and third trimester to generate a normative transcriptome based on sex and gestation. STUDY DESIGN: We analyzed 124 first trimester (T1, 59 female and 65 male) and 43 third trimester (T3, 18 female and 25 male) samples for sex differences within each trimester and sex-specific gestational differences. RESULTS: Placenta shows more significant sexual dimorphism in T1, with 94 T1 and 26 T3 differentially expressed genes (DEGs). The sex chromosomes contributed 60.6% of DEGs in T1 and 80.8% of DEGs in T3, excluding X/Y pseudoautosomal regions. There were 6 DEGs from the pseudoautosomal regions, only significant in T1 and all upregulated in males. The distribution of DEGs on the X chromosome suggests genes on Xp (the short arm) may be particularly important in placental sex differences. Dosage compensation analysis of X/Y homolog genes shows expression is primarily contributed by the X chromosome. In sex-specific analyses of first versus third trimester, there were 2815 DEGs common to both sexes upregulated in T1, and 3263 common DEGs upregulated in T3. There were 7 female-exclusive DEGs upregulated in T1, 15 female-exclusive DEGs upregulated in T3, 10 male-exclusive DEGs upregulated in T1, and 20 male-exclusive DEGs upregulated in T3. DISCUSSION: This is the largest cohort of placentas across gestation from healthy pregnancies defining the normative sex dimorphic gene expression and sex common, sex specific and sex exclusive gene expression across gestation. The first trimester has the most sexually dimorphic transcripts, and the majority were upregulated in females compared to males in both trimesters. The short arm of the X chromosome and the pseudoautosomal region is particularly critical in defining sex differences in the first trimester placenta. As pregnancy is a dynamic state, sex specific DEGs across gestation may contribute to sex dimorphic changes in overall outcomes.

Labour and premature delivery differentially affect the expression of the endocannabinoid system in the human placenta.

Plasma concentrations of N-arachidonyletholamine (AEA), N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA) increase at term and can predict when a woman is likely to go into labour. We hypothesised that increased plasma AEA concentrations in women in preterm and term labour might also be increased and have a function in the placenta at the end of pregnancy. Here we examined the expression of the N-acylethanolamine-modulating enzymes fatty acid amide hydrolase (FAAH) and N-acyl-phosphatidylethanolamine-specific phospholipase-D (NAPE-PLD) and of the cannabinoid receptors (CB1 and CB2) in the placenta and their activation in an in vitro model of the third-trimester placenta to determine if those expressions change with labour and have functional significance. Expression of CB1, CB2, FAAH and NAPE-PLD was examined by immunohistochemistry (IHC) and RT-qPCR in placental samples obtained from four patient groups: preterm not in labour (PTNL), term not in labour (TNL), preterm in labour (PTL) and term in labour (TL). Additionally, the effects of AEA on a third-trimester human cell line (TCL-1) were evaluated. All ECS components were present in the third-trimester placenta, with NAPE-PLD and CB2 being the key modulated proteins in terms of expression. Functionally, AEA reduced TCL-1 cell numbers through the actions of the CB2 receptor whilst CB1 maintained placental integrity through the expression of the transcription regulators histone deacetylase 3, thyroid hormone receptor ß 1 and the modulation of 5α reductase type 1. The placenta in the third trimester and at term is different from the placenta in the first trimester with respect to the expression of CB1, CB2, FAAH and NAPE-PLD, and the expression of these proteins is affected by labour. These data suggest that early perturbation of some ECS components in the placenta may cause AEA-induced PTL and thus PTB.

WNK1 mediates amphiregulin-induced MMP9 expression and cell invasion in human extravillous trophoblast cells.

The invasion of human extravillous trophoblast (EVT) cells is a critical event required for a successful pregnancy. Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), has been shown to stimulate cell invasion in an immortalized human EVT cell line, HTR-8/SVneo. The with-no-lysine kinase 1 (WNK1) is involved in regulating cell invasion. It is known that WNK1 is expressed in the human placenta, but its role in human EVT cells remains unknown. In the present study, we show that AREG treatment phosphorylated WNK1 at Thr60 in both HTR-8/SVneo and primary human EVT cells. The stimulatory effect of AREG on WNK1 phosphorylation was mediated by the activation of PI3K/AKT, but not the ERK1/2 signaling pathway. AREG upregulated matrix metalloproteinase 9 (MMP9) but not MMP2. In addition, cell invasiveness was increased in response to the treatment of AREG. Using the siRNA-mediated knockdown approach, our results showed that the knockdown of WNK1 attenuated the AREG-induced upregulation of MMP9 expression and cell invasion. Moreover, the expression of WNK1 was downregulated in the placentas with preeclampsia, a disease resulting from insufficiency of EVT cell invasion during pregnancy. This study discovers the physiological function of WNK1 in human EVT cells and provides important insights into the regulation of MMP9 and cell invasion in human EVT cells.

A spatially resolved timeline of the human maternal-fetal interface.

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5 and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

The effects of gestational diabetes mellitus with maternal age between 35 and 40 years on the metabolite profiles of plasma and urine.

BACKGROUND: Gestational diabetes mellitus (GDM) is defined as impaired glucose tolerance in pregnancy and without a history of diabetes mellitus. While there are limited metabolomic studies involving advanced maternal age in China, we aim to investigate the metabolomic profiling of plasma and urine in pregnancies complicated with GDM aged at 35-40 years at early and late gestation. METHODS: Twenty normal and 20 GDM pregnant participants (≥ 35 years old) were enlisted from the Complex Lipids in Mothers and Babies (CLIMB) study. Maternal plasma and urine collected at the first and third trimester were detected using gas chromatography-mass spectrometry (GC-MS). RESULTS: One hundred sixty-five metabolites and 192 metabolites were found in plasma and urine respectively. Urine metabolomic profiles were incapable to distinguish GDM from controls, in comparison, there were 14 and 39 significantly different plasma metabolites between the two groups in first and third trimester respectively. Especially, by integrating seven metabolites including cysteine, malonic acid, alanine, 11,14-eicosadienoic acid, stearic acid, arachidic acid, and 2-methyloctadecanoic acid using multivariant receiver operating characteristic models, we were capable of discriminating GDM from normal pregnancies with an area under curve of 0.928 at first trimester. CONCLUSION: This study explores metabolomic profiles between GDM and normal pregnancies at the age of 35-40 years longitudinally. Several compounds have the potential to be biomarkers to predict GDM with advanced maternal age. Moreover, the discordant metabolome profiles between the two groups could be useful to understand the etiology of GDM with advanced maternal age.

Proteome-defined changes in cellular pathways for decidua and trophoblast tissues associated with location and viability of early-stage pregnancy.

BACKGROUND: In early pregnancy, differentiating between a normal intrauterine pregnancy (IUP) and abnormal gestations including early pregnancy loss (EPL) or ectopic pregnancy (EP) is a major clinical challenge when ultrasound is not yet diagnostic. Clinical treatments for these outcomes are drastically different making early, accurate diagnosis imperative. Hence, a greater understanding of the biological mechanisms involved in these early pregnancy complications could lead to new molecular diagnostics. METHODS: Trophoblast and endometrial tissue was collected from consenting women having an IUP (n = 4), EPL (n = 4), or EP (n = 2). Samples were analyzed by LC-MS/MS followed by a label-free proteomics analysis in an exploratory study. For each tissue type, pairwise comparisons of different pregnancy outcomes (EPL vs. IUP and EP vs. IUP) were performed, and protein changes having a fold change ≥ 3 and a Student's t-test p-value ≤ 0.05 were defined as significant. Pathway and network classification tools were used to group significantly changing proteins based on their functional similarities. RESULTS: A total of 4792 and 4757 proteins were identified in decidua and trophoblast proteomes. For decidua, 125 protein levels (2.6% of the proteome) were significantly different between EP and IUP, whereas EPL and IUP decidua were more similar with only 68 (1.4%) differences. For trophoblasts, there were 66 (1.4%) differences between EPL and IUP. However, the largest group of 344 differences (7.2%) was observed between EP and IUP trophoblasts. In both tissues, proteins associated with ECM remodeling, cell adhesion and metabolic pathways showed decreases in EP specimens compared with IUP and EPL. In trophoblasts, EP showed elevation of inflammatory and immune response pathways. CONCLUSIONS: Overall, differences between an EP and IUP are greater than the changes observed when comparing ongoing IUP and nonviable intrauterine pregnancies (EPL) in both decidua and trophoblast proteomes. Furthermore, differences between EP and IUP were much higher in the trophoblast than in the decidua. This observation is true for the total number of protein changes as well as the extent of changes in upstream regulators and related pathways. This suggests that biomarkers and mechanisms of trophoblast function may be the best predictors of early pregnancy location and viability.

α-tocopherol prevents oxidative stress-induced proliferative dysfunction in first-trimester human placental (HTR-8/SVneo) cells.

Extravillous trophoblasts (EVTs) are the main participants in the process of placentation, an early process critical for placental growth and function involving an adequate invasion and complete remodelling of the maternal spiral arteries during early pregnancy. An increase in oxidative stress during pregnancy is associated with the onset and progression of several pregnancy disorders, including preeclampsia and gestational diabetes mellitus and it also occurs due to exposure of pregnant women to some xenobiotics (eg. alcohol). This study aimed to investigate how oxidative stress affects EVTs, and the ability of several distinct antioxidant agents to prevent these changes. For this, we exposed HTR8/SVneo cells to tert-butylhydroperoxide (0.5 µM; 24 h), which was able to increase lipid peroxidation and protein carbonyl levels. Under these conditions, there was a decrease in proliferation rates, culture growth, migratory and angiogenic capacities and an increase in the apoptosis rates. The antiproliferative effect of TBH was supressed by simultaneous treatment of the cells with α-tocopherol, but other antioxidants (vitamin C, allopurinol, apocynin, N-acetylcysteine, quercetin and resveratrol) were ineffective. α-tocopherol was also able to abolish the effect of TBH on lipid peroxidation and protein carbonyl levels. Overall, our results show that oxidative stress interferes with EVT characteristics essential for the placentation process, which may contribute to the association between oxidative stress and pregnancy disorders. Our results also show that the nature of the in vitro model of oxidative stress-induction is an important determinant of the cellular consequences of oxidative stress and, therefore, of the efficacy of antioxidants.

A Prospective Study of Early-pregnancy Thyroid Markers, Lipid Species, and Risk of Gestational Diabetes Mellitus.

CONTEXT: While the associations between thyroid markers and gestational diabetes mellitus (GDM) have been extensively studied, the results are inconclusive and the mechanisms remain unclear. OBJECTIVE: We aimed to investigate the prospective associations of thyroid markers in early gestation with GDM risk, and examine the mediating effects through lipid species. METHODS: This study included 6068 pregnant women from the Tongji-Shuangliu Birth Cohort. Maternal serum thyroid markers (free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone, thyroid peroxidase antibody, and thyroglobulin antibody) were measured before 15 weeks. Deiodinase activity was assessed by fT3/fT4 ratio. Plasma lipidome were quantified in a subset of 883 participants. RESULTS: Mean age of the participants was 26.6 ± 3.7 years, and mean gestational age was 10.3 ± 2.0 weeks. Higher levels of fT4 were associated with a decreased risk of GDM (OR = 0.73 comparing the extreme quartiles; 95% CI 0.54, 0.98, Ptrend = .043), while higher fT3/fT4 ratio was associated with an increased risk of GDM (OR = 1.43 comparing the extreme quartiles; 95% CI 1.06, 1.93, Ptrend = .010) after adjusting for potential confounders. Multiple linear regression suggested that fT3/fT4 ratio was positively associated with alkylphosphatidylcholine 36:1, phosphatidylethanolamine plasmalogen 38:6, diacylglyceride 18:0/18:1, sphingomyelin 34:1, and phosphatidylcholine 40:7 (false discovery rate [FDR] adjusted P < .05). Mediation analysis indicated 67.9% of the association between fT3/fT4 ratio and GDM might be mediated through the composite effect of these lipids. CONCLUSION: Lower concentration of serum fT4 or higher fT3/fT4 ratio in early pregnancy was associated with an increased risk of GDM. The association of fT3/fT4 ratio with GDM was largely mediated by specific lipid species.

Fragile X-Related Protein 1 (FXR1) Promotes Trophoblast Migration at Early Pregnancy via Downregulation of GDF-15 Expression.

Fragile X-related protein 1 (FXR1) is an RNA-binding protein that can regulate specific mRNA decay in cells. Our previous study showed that FXR1 expression was significantly decreased in trophoblasts from patients with unexplained recurrent spontaneous abortion (RSA); however, the role of FXR1 in trophoblast function during early placenta development has not been fully elucidated. In this study, we found that knockdown of FXR1 using siRNA effectively inhibited the migration of HTR-8 cells and extravillous trophoblast (EVT) outgrowth in an ex vivo extravillous explant culture model. Furthermore, through analysis of a panel of cytokines, we found that the GDF-15 protein was upregulated after knockdown of FXR1 in HTR-8/SVneo cells. This was further confirmed by western blotting and immunofluorescence in HTR-8/SVneo cells and an extravillous explant. Our data also showed that FXR1 expression was downregulated and GDF-15 was upregulated in chorionic villous tissues from RSA patients compared with those from healthy controls (HCs). Further, immunohistochemistry showed a strong expression of GDF-15 in chorionic villous tissue in the RSA group, which was mainly distributed in villous trophoblasts (CTBs) and syncytiotrophoblasts (STBs). Moreover, knockdown of GDF-15 enhanced the migration of HTR-8 cells, while overexpression of GDF-15 using plasmid or treatment with recombinant human GDF-15 protein inhibited trophoblast migration. Importantly, RNA-binding protein immunoprecipitation showed that FXR1 directly bound to the 3'-UTR of GDF-15 mRNA to promote GDF-15 mRNA decay. Together, our data provide new insight into the function of FXR1 in human placenta via regulation of GDF-15 expression in trophoblasts and suggest a possible pathological process involved in RSA.

Functional Analysis of p21Cip1/CDKN1A and Its Family Members in Trophoblastic Cells of the Placenta and Its Roles in Preeclampsia.

Preeclampsia (PE), a gestational hypertensive disease originating from the placenta, is characterized by an imbalance of various cellular processes. The cell cycle regulator p21Cip1/CDKN1A (p21) and its family members p27 and p57 regulate signaling pathways fundamental to placental development. The aim of the present study was to enlighten the individual roles of these cell cycle regulators in placental development and their molecular involvement in the pathogenesis of PE. The expression and localization of p21, phospho-p21 (Thr-145), p27, and p57 was immunohistochemically analyzed in placental tissues from patients with early-onset PE, early-onset PE complicated by the HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome as well as late-onset PE compared to their corresponding control tissues from well-matched women undergoing caesarean sections. The gene level was evaluated using real-time quantitative PCR. We demonstrate that the delivery mode strongly influenced placental gene expression, especially for CDKN1A (p21) and CDKN1B (p27), which were significantly upregulated in response to labor. Cell cycle regulators were highly expressed in first trimester placentas and impacted by hypoxic conditions. In support of these observations, p21 protein was abundant in trophoblast organoids and hypoxia reduced its gene expression. Microarray analysis of the trophoblastic BeWo cell line depleted of p21 revealed various interesting candidate genes and signaling pathways for the fusion process. The level of p21 was reduced in fusing cytotrophoblasts in early-onset PE placentas and depletion of p21 led to reduced expression of fusion-related genes such as syncytin-2 and human chorionic gonadotropin (ß-hCG), which adversely affected the fusion capability of trophoblastic cells. These data highlight that cell cycle regulators are important for the development of the placenta. Interfering with p21 influences multiple pathways related to the pathogenesis of PE.

Hypoxia-induced DEC1 mediates trophoblast cell proliferation and migration via HIF1α signaling pathway.

In early pregnancy, hypoxia is a typical extrinsic factor that regulates EVT functions including proliferation, migration and invasion which are essential for a successful pregnancy. Human differentiated embryonic chondrocyte-expressed gene 1 (DEC1), a hypoxia-regulated gene, has been reported to be overexpressed in several types of cancers. Given that the placenta and the cancer share several similarities with respect to their capacity to proliferate and invade adjacent tissues, we focused on the role of DEC1 on trophoblast function in a physiologically hypoxic environment, which may be associated with unexplained recurrent spontaneous abortion (URSA).In our study, we measured the expression of HIF-1α and DEC1 in first-trimester villi through real-time-PCR (RT-PCR) and immunohistochemical analysis. in vitro, DEC1 expression was downregulated in trophoblast cells via DEC1-specific shRNA plasmid transfection. The expression of DEC1 and HIF-1α was detected via western blotting and RT-PCR analysis. The proliferation and migration of HTR-8/SVneo cells were assayed using CCK-8 and Transwell migration assays, respectively.Our results indicated that the expression of DEC1 was significantly reduced in villi of URSA compared to that in normal pregnant women. in vitro, hypoxia induced the expression of HIF-1ɑ and DEC1 and upregulated proliferation and migration of the HTR-8/SVneo cells. Knockdown of DEC1 inhibited proliferation and migration of HTR-8/SVneo cells exposure to hypoxia. Furthermore, inhibition of HIF1α expression resulted in a significant decrease in DEC1. These findings illustrate that hypoxia-induced DEC1 expression promotes trophoblast cell proliferation and migration through the HIF1α signaling pathway, which plays an important role during placentation.

The interactive effects of non-alcoholic fatty liver disease and hemoglobin concentration in the first trimester on the development of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is associated with adverse perinatal and maternal outcomes. Epidemiological studies have reported that non-alcoholic fatty liver disease (NAFLD) and a high hemoglobin (Hb) concentration are risk factors for GDM in the middle trimester. However, no consistent conclusions have been reached, especially in Chinese pregnant women. A case-control study was conducted to better understand the associations between NAFLD and Hb concentration in the first trimester and the risk of GDM and their interactive effects. Multivariable logistic regression analysis and a cross-product term of Hb and steatosis were used to evaluate the associations between first trimester Hb concentration, steatosis, and GDM and their interactive effects. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using two-sided statistical tests at an alpha level of 0.05. For the study, 1,017 normal pregnant women, and 343 pregnant women diagnosed with GDM (25.22%) were recruited from the First Hospital of Shanxi Medical University, Shanxi Province, China. NAFLD-associated steatosis was found to be independent risk factors for developing GDM compared with grade 0 steatosis, with ORs of 1.98 (95% CI: 1.35-2.89) and 2.27 (95% CI:1.29-3.96), respectively. Meanwhile, a high Hb concentration was found to be a risk factor for developing GDM compared with the normal Hb concentration (OR = 1.88; 95% CI:1.24-2.83). The risk of developing GDM was more pronounced among pregnant women who had both high-grade steatosis and higher Hb concentrations during their first trimester (OR = 6.24; 95% CI: 1.81-23.66). However, we found no significant interactions between Hb concentration and steatosis grade. In conclusion, our study confirmed that a high Hb concentration and NAFLD-associated steatosis during the first trimester play important roles in predicting the risk of GDM in Chinese women. Future studies are required to verify the interactive effects between NAFLD-associated steatosis and Hb concentration.

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.

Low fetal fraction in obese women at first trimester cell-free DNA based prenatal screening is not accompanied by differences in total cell-free DNA.

OBJECTIVE: Reasons for first trimester noninvasive prenatal screening (NIPS) test failure in obese women remain elusive. As dilution from maternal sources may be explanatory, we determined the relationship between obesity, fetal fraction (FF), and total cell-free DNA (cfDNA) using our NIPS platform. METHODS: We assessed differences in first trimester (≤14 weeks) FF, indeterminate rate, and total cfDNA between obese (n = 518) and normal-weight women (n = 237) after exclusion of confounders (anticoagulation, autoimmunity, aneuploidy) and controlling for covariates. RESULTS: Fetal fraction was lower, and the indeterminate rate higher, in obese compared to controls (9.2% ± 4.4 vs. 12.5% ± 4.5, p < 0.001 and 8.4 vs. 1.7%, p < 0.001, respectively), but total cfDNA was not different (92.0 vs. 82.1 pg/µl, p = 0.10). For each week, the FF remained lower in obese women (all p < 0.01) but did not increase across the first trimester for either group. Obesity increased the likelihood of indeterminate result (OR 6.1, 95% CI 2.5, 14.8; p < 0.001) and maternal body mass index correlated with FF (ß -0.27, 95% CI -0.3, -0.22; p < 0.001), but not with total cfDNA (ß 0.49, 95% CI -0.55, 1.53; p = 0.3). CONCLUSIONS: First trimester obese women have persistently low FF and higher indeterminate rates, without differences in total cfDNA, suggesting placental-specific mechanisms versus dilution from maternal sources as a potential etiology.

Vaginal metabolic profiles during pregnancy: Changes between first and second trimester.

During pregnancy, the vaginal microbiome plays an important role in both maternal and neonatal health outcomes. Throughout pregnancy, the vaginal microbial composition undergoes significant changes, including a decrease in overall diversity and enrichment with Lactobacillus spp. In turn, the modifications in the microbial profiles are associated with shifts in the composition of vaginal metabolites. In this study, we characterized the vaginal metabolic profiles throughout pregnancy at two different gestational ages, correlating them with a microscopic evaluation of the vaginal bacterial composition. A total of 67 Caucasian pregnant women presenting to the Family Advisory Health Centres of Ravenna (Italy) were enrolled and a vaginal swab was collected at gestational ages 9-13 weeks (first trimester) and 20-24 weeks (second trimester). The composition of the vaginal microbiome was assessed by Nugent score and women were divided in 'H' (normal lactobacilli-dominated microbiota), 'I' (intermediate microbiota), and 'BV' (bacterial vaginosis) groups. Starting from the cell-free supernatants of the vaginal swabs, a metabolomic analysis was performed by means of a 1H-NMR spectroscopy. From the first to the second trimester, a greater number of women showed a normal lactobacilli-dominated microbiota, with a reduction of cases of dysbiosis. These microbial shifts were associated with profound changes in the vaginal metabolic profiles. Over the weeks, a significant reduction in the levels of BV-associated metabolites (e.g. acetate, propionate, tyramine, methylamine, putrescine) was observed. At the same time, the vaginal metabolome was characterized by higher concentrations of lactate and of several amino acids (e.g. tryptophan, threonine, isoleucine, leucine), typically found in healthy vaginal conditions. Over time, the vaginal metabolome became less diverse and more homogeneous: in the second trimester, women with BV showed metabolic profiles more similar to the healthy/intermediate groups, compared to the first trimester. Our data could help unravel the role of vaginal metabolites in the pathophysiology of pregnancy.

Age and Sex-Related Changes in Human First-Trimester Placenta Transcriptome and Insights into Adaptative Responses to Increased Oxygen.

Physiological oxygen tension rises dramatically in the placenta between 8 and 14 weeks of gestation. Abnormalities in this period can lead to gestational diseases, whose underlying mechanisms remain unclear. We explored the changes at mRNA level by comparing the transcriptomes of human placentas at 8-10 gestational weeks and 12-14 gestational weeks. A total of 20 samples were collected and divided equally into four groups based on sex and age. Cytotrophoblasts were isolated and sequenced using RNAseq. Key genes were identified using two different methods: DESeq2 and weighted gene co-expression network analysis (WGCNA). We also constructed a local database of known targets of hypoxia-inducible factor (HIF) subunits, alpha and beta, to investigate expression patterns likely linked with changes in oxygen. Patterns of gene enrichment in and among the four groups were analyzed based on annotations of gene ontology (GO) and KEGG pathways. We characterized the similarities and differences between the enrichment patterns revealed by the two methods and the two conditions (age and sex), as well as those associated with HIF targets. Our results provide a broad perspective of the processes that are active in cytotrophoblasts during the rise in physiological oxygen, which should benefit efforts to discover possible drug-targeted genes or pathways in the human placenta.

Decidual-derived RANKL facilitates macrophages accumulation and residence at the maternal-fetal interface in human early pregnancy.

PROBLEM: During the first trimester, the accumulation of macrophages, which is the second largest decidual leukocyte population (~20%) at the maternal-fetal interface, is quite vital for a successful pregnancy, including embryo implantation, trophoblast invasion, and vascular remodeling. The mechanism of the enrichment and redistribution of macrophages in the uterine decidua of early pregnancy is largely unclear. METHOD OF STUDY: A total of 37 women with normal early pregnancies were included. Primary decidual macrophages (dMφs) (n = 37) and primary decidual stromal cells (DSCs) (n = 37) were isolated, and the adhesion molecules were analyzed by flow cytometry (FCM). Adhesive experiment was carried out to evaluate the adhesion capacity by counting cell numbers of dMφs adhered to DSCs in a co-culture system. RESULTS: We found that RANK+ dMφs was the dominating subtype at the maternal-fetal interface. The expression of adhesion molecules (eg, CD29, CD31, CD54, and CD62L) on the surface of RANK+ dMφs was higher than that of RANK- dMφs. After co-culture with DSCs, the expression of adhesion molecules on dMφs was up-regulated in a RANKL-dependent manner. Meanwhile, dMφs promoted the releasing of RANKL on DSCs after co-culture. Consistently, dMφs exhibited the lessoned capacity of adhesion to DSCs when blocking the crosstalk of RANKL-RANK between the DSCs and dMφs in vitro. CONCLUSION: These results suggest that the interaction of RANKL-RANK up-regulates the expression of adhesion molecules on the surface of dMφs, contributing to the accumulation and residence of dMφs in human early pregnancy.

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells.

Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.