Trimester Placenta Research Articles

Trophoblast H2S Maintains Early Pregnancy via Regulating Maternal-Fetal Interface Immune Hemostasis.

Dysregulated immune hemostasis occurs in unexplained recurrent spontaneous abortion (URSA). Synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), hydrogen sulfide (H2S) promotes regulatory T-cell differentiation and regulates immune hemostasis; yet, its role in URSA is elusive. To determine if H2S plays a role in early pregnancy and if dysregulated H2S signaling results in recurrent spontaneous abortion. First trimester placenta villi and decidua were collected from normal and URSA pregnancies. Protein expression was examined by immunohistochemistry and immunoblotting. Human trophoblast HTR8/SVneo and JEG3 cells were treated with H2S donors; HTR8/SVneo cells were transfected with CBS ribonucleic acid interference (RNAi) or complementary deoxyribonucleic acid. Cell migration and invasion were determined by transwell assays; trophoblast transcriptomes were determined by RNA sequencing (RNA-seq). Wild-type, CBS-deficient, and CBA/J × DBA/2 mice were treated with CBS and CSE inhibitors or H2S donors to determine the role of H2S in early pregnancy in vivo. CBS and CSE proteins showed cell-specific expressions, but only CBS decreased in the villous cytotrophoblast in URSA versus normal participants. H2S donors promoted migration and invasion and MMP-2 and VEGF expression in human placenta trophoblast cells that contain SV40 viral deoxyribonucleic acid sequences (HTR8/SVneo) and human placenta trophoblast cells (JEG3 cells), similar to forced CBS expression in HTR8/SVneo cells. The CBS-responsive transcriptomes in HTR8/SVneo cells contained differentially regulated genes (ie, interleukin-1 receptor and prostaglandin-endoperoxide synthase 2) that are associated with nuclear factor-κB-mediated inflammatory response. In vivo, dysregulated CBS/H2S signaling significantly increased embryonic resorption and decidual T-helper 1/T-helper 2 imbalance in mice, which was partially rescued by H2S donors. CBS/H2S signaling maintains early pregnancy, possibly via regulating maternal-fetal interface immune hemostasis, offering opportunities for H2S-based immunotherapies for URSA.

NPFF increases fusogenic proteins syncytin 1 and syncytin 2 via GCM1 in first trimester primary human cytotrophoblast cells.

Neuropeptide FF (NPFF) is well-known for its roles in the central nervous system. Despite studies demonstrating that NPFF receptor 2 (NPFFR2) mRNA is highest in placenta, nothing is known about NPFF-NPFFR2 functions in placental development. Here, we investigated the effects of NPFF-NPFFR2 on expression of syncytial [human chorionic gonadotropin (hCG) β] and fusogenic [syncytin 1, syncytin 2, and glial cells missing 1 (GCM1)] genes in first trimester primary human cytotrophoblast cells. By analyzing two publicly available microarray data sets, we found that NPFF is consistently expressed throughout gestation whereas NPFFR2 increases in first trimester and is elevated in placenta samples from women with preeclampsia. Immunohistochemistry showed that NPFFR2, syncytin 1/2, and GCM1 each displayed unique patterns of expression among different trophoblast populations in first trimester placenta. Treatment of primary human cytotrophoblast cells with NPFF increased the mRNA and protein levels of hCG β, syncytin 1, syncytin 2, and GCM1; and knockdown of NPFFR2 abolished these effects. Interestingly, GCM1 mediated NPFF-induced upregulation of syncytin 1 and syncytin 2, but not hCG β, in primary human cytotrophoblasts. Our results demonstrate that NPFF acts via NPFFR2 to enhance production of hCG β and promote GCM1-dependent expression of syncytin 1 and 2 in human cytotrophoblasts.

Placental Pathology in COVID-19.

ObjectivesTo describe histopathologic findings in the placentas of women with coronavirus disease 2019 (COVID-19) during pregnancy.MethodsPregnant women with COVID-19 delivering between March 18, 2020, and May 5, 2020, were identified. Placentas were examined and compared to historical controls and women with placental evaluation for a history of melanoma.ResultsSixteen placentas from patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were examined (15 with live birth in the third trimester, 1 delivered in the second trimester after intrauterine fetal demise). Compared to controls, third trimester placentas were significantly more likely to show at least one feature of maternal vascular malperfusion (MVM), particularly abnormal or injured maternal vessels, and intervillous thrombi. Rates of acute and chronic inflammation were not increased.The placenta from the patient with intrauterine fetal demise showed villous edema and a retroplacental hematoma.ConclusionsRelative to controls, COVID-19 placentas show increased prevalence of decidual arteriopathy and other features of MVM, a pattern of placental injury reflecting abnormalities in oxygenation within the intervillous space associated with adverse perinatal outcomes. Only 1 COVID-19 patient was hypertensive despite the association of MVM with hypertensive disorders and preeclampsia. These changes may reflect a systemic inflammatory or hypercoagulable state influencing placental physiology.

Spliceosome protein EFTUD2 is upregulated in the trophoblast of spontaneous miscarriage and hydatidiform mole

BackgroundElongation factor Tu GTP binding domain containing 2 (EFTUD2) is an alternative splicing factor that modulates cell differentiation and activation processes. EFTUD2 is known to modulate immune responses and mutation of the EFTUD2-gene lead to fetal malformation. Little is known about its expression and role in normal and disturbed first trimester pregnancy. Patients and methodsWe investigated the expression of EFTUD2 in placental tissue obtained from patients with normal (n = 14), spontaneous miscarriage (n = 15) and molar (n = 14) pregnancy by immunohistochemistry. The expression of EFTUD2 was correlated on the protein level with known immune modulatory proteins like pregnancy zone protein (PZP) and in addition with human chorionic gonadotropin (hCG). Furthermore, we analysed the EFTUD2 and PZP expression in vitro after stimulation of the chorioncarcinoma cell line JEG-3 with hCG. ResultsEFTUD2 is significantly upregulated in the syncytiotrophoblast of spontaneous miscarriage (p = 0.003) and molar pregnancy (p = 0.003) compared to week of gestation-adjusted normal first trimester placentas. PZP is negatively correlated (p = 0.021) to EFTUD2 in the syncytiotrophoblast and is therefore significantly downregulated in miscarriage (p = 0.028) and mole pregnancy (p = 0.006). In addition, hCG is positively correlated to EFTUD2 in mole pregnancy. The addition of hCG to chorioncarcinoma cell lines JEG-3 in vitro stimulated EFTUD2 expression in these cells (p = 0.027). ConclusionRegulation of alternative splicing seems crucial for a successful ongoing pregnancy. The up-regulated elongation factor EFTUD2 may have a critical role in miscarriage.

OR24-07 Fetal Sex Impacts First Trimester Maternal-Fetal Communication in Humans

The placenta serves as a regulator of fetal growth throughout pregnancy. Signaling at the maternal-fetal interface is critical during placentation and lays the groundwork for placenta function, affecting pregnancy outcomes. Fetal growth is impacted by fetal sex, with males larger than females, and maternal gestational diabetes and obesity independently increase the risk of macrosomia in male fetuses only. We previously demonstrated differentially expressed genes (DEGs) among sexes involves ancient canonical pathways and metabolic functions in placenta tissue. As these are likely impacted by signaling at the maternal-fetal interface, our aim here was to identify sex differences in signaling at the maternal-fetal interface and among individual cell types within the placenta to explain these differences. RNA-sequencing of first trimester placenta and maternal decidua as well as single cell RNA-sequencing in first trimester placenta was performed in ongoing pregnancies. We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. From these, 35 of 115 receptors and/or ligand genes were also found to be upstream regulators of pathways critical in sexually dimorphic placentation which may define regulation. Single cell analysis identified five major cell types (trophoblasts, stromal cells, hofbauer cells, antigen presenting cells, and endothelial cells), and all had sexually dimorphic genes. Among individual cell types, ligands from the CC-family of cytokines were most highly representative in females, with their corresponding receptors present on the maternal surface. Furthermore, upstream regulator analysis of sexually dimorphic genes demonstrated TGFβ1 and estradiol to significantly affect all cell types. Dihydrotestosterone, which is produced by the male fetus, was an upstream regulator that was most significant for the trophoblast population. In addition, gene ontology enrichment analysis identified distinctive enriched functions between male and female trophoblasts, with cytokine mediated signaling pathways most representative. MUC15 and NOTUM were the most highly expressed sexually dimorphic autosomal genes found in distinct cell types of the trophoblast population, cell types critical for placentation and nutrient exchange. Thus, differences in hormone and immune signaling pathways may account for differential gene expression and differences in trophoblast function during placentation, which may in turn explain developmental differences, including fetal size, well-being, and overall outcomes.

Human Placenta Harbors a Diverse Immune Landscape With Inflammatory Potential

Throughout pregnancy, the maternal immunity is tolerant to the developing fetus. However, recent work suggests that the fetal immune system in utero is highly developed and may also contribute to tolerance. We hypothesized that each layer (maternal decidua, fetal membranes, and fetal villi) of the second trimester (ST) placenta harbors a distinct immune landscape. Mass cytometry of 11 ST placentas demonstrated that each tissue did have a unique immune profile. Specifically, the villi contain chemokine receptor (CCR)lo innate cells and enrichment of macrophages and DCs. In contrast, the decidua’s innate cells were CCRhi with abundant NK cells. Random forest classification accurately assigned samples as villi or decidua based on three distinct populations. Memory CD8 T cells were the dominant adaptive cells in all three layers and were more activated in decidua than villi. However, T cells within the villi could secrete TNFα/IFNγ when stimulated with maternal components, suggesting that villi T cells become proinflammatory upon exposure to maternal antigens. Furthermore, T cells from preterm villi had more activation transcripts than ST and term villi. Collectively, these findings illustrate a complex arsenal of immune cells within the placenta in utero and suggest that fetal T cells may contribute to preterm pathology.

Second and third trimester fetal ultrasound population screening for risks of preterm birth and small-size and large-size for gestational age at birth: a population-based prospective cohort study

BackgroundPreterm birth, small size for gestational age (SGA) and large size for gestational age (LGA) at birth are major risk factors for neonatal and long-term morbidity and mortality. It is unclear which periods of pregnancy are optimal for ultrasound screening to identify fetuses at risk of preterm birth, SGA or LGA at birth. We aimed to examine whether single or combined second and third trimester ultrasound in addition to maternal characteristics at the start of pregnancy are optimal to detect fetuses at risk for preterm birth, SGA and LGA.MethodsIn a prospective population-based cohort among 7677 pregnant women, we measured second and third trimester estimated fetal weight (EFW), and uterine artery pulsatility and umbilical artery resistance indices as placenta flow measures. Screen positive was considered as EFW or placenta flow measure < 10th or > 90th percentile. Information about maternal age, body mass index, ethnicity, parity, smoking, fetal sex and birth outcomes was available from questionnaires and medical records. Screening performance was assessed via receiver operating characteristic (ROC) curves and area under the curve (AUC) along with sensitivity at different false-positive rates.ResultsMaternal characteristics only and in combination with second trimester EFW had a moderate performance for screening for each adverse birth outcome. Screening performance improved by adding third trimester EFW to the maternal characteristics (AUCs for preterm birth 0.64 (95%CI 0.61 to 0.67); SGA 0.79 (95%CI 0.78 to 0.81); LGA 0.76 (95%CI 0.75; 0.78)). Adding third trimester placenta measures to this model improved only screening for risk of preterm birth (AUC 0.72 (95%CI 0.66 to 0.77) with sensitivity 37% at specificity 90%) and SGA (AUC 0.83 (95%CI 0.81 to 0.86) with sensitivity 55% at specificity 90%). Combining second and third trimester fetal and placental ultrasound did not lead to a better performance as compared to using only third trimester results.ConclusionsCombining single third trimester fetal and placental ultrasound results with maternal characteristics has the best screening performance for risks of preterm birth, SGA and LGA. As compared to second trimester screening, third trimester screening may double the detection of fetuses at risk of common adverse birth outcomes.

Feasibility of image registration and fusion for evaluation of structure and perfusion of the entire second trimester placenta by three-dimensional power Doppler ultrasound

BackgroundPlacental perfusion can be evaluated by 3D power Doppler ultrasound (3D PD-US), particularly using the validated tool 3D Fractional Moving Blood Volume (3D-FMBV); however regional variability and size limitations beyond the first trimester mean that multiple 3D PD-US volumes are required to evaluate the whole organ. PurposeWe assessed the feasibility of manual offline stitching of second trimester 3D PD-US volumes of the placenta to assess whole organ perfusion using 3D-FMBV. Materials and methodsThis was a single-centre, prospective, observational cohort study of 36 normal second trimester singleton pregnancies with anterior placentas. 3D PD-US placental volumes were manually segmented offline and stitched together by rigid registration using manually selected, pair-wise coordinates. Data acquisition and offline volume segmentation and stitching were triplicated by a single observer with Dice similarity coefficient (DSC) and Hausdorff distance used to assess consistency. Intraclass correlation coefficient (ICC) was used to assess intra-observer repeatability of 3D-FMBV and placental volume. ResultsAcquisition and stitching success were 94% and 88%, respectively. Median time for acquisition, segmentation and stitching were 13 min, 40 min and 95 min, respectively. Median intra-observer DSCs were 0.94 and 0.88, and Hausdorff distances were 11.85 mm and 36.6 mm, for segmentations and stitching, respectively. Conclusion3D-ultrasound volume stitching of the placenta is technically feasible. Intra-observer repeatability was good to excellent for all measured parameters. This work demonstrates technical feasibility; further studies may provide the basis of an in-vivo assessment tool to measure the placenta in mid-to late pregnancy.

The Differential Expression of ERAP1/ERAP2 and Immune Cell Activation in Pre-eclampsia.

Pre-eclampsia (PE) is a disorder of pregnancy, often leading to serious and fatal complications. Endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1/ERAP2) are present in the placenta. They are involved in processes regulating blood pressure, angiogenesis, cytokine receptor shedding, and immune recognition. Previous studies have associated both ERAP1/ERAP2 genetic variants with PE, although the underlying mechanisms remain unknown. Less is known about the roles for these enzymes in early placentation, which could be a contributory factor to PE. To ascertain whether ERAP1/ERAP2 change in PE and whether such a change is present before PE is clinically diagnosed, we analyzed mRNA and ERAP1/2 protein expression in the placenta in the early first trimester (8–14 weeks) and at delivery in normotensive or PE women (n = 12/group). Gene expression was analyzed using qPCR, and protein expression and localization were assessed by immunohistochemistry. Additionally, we profiled peripheral immune cells from normotensive and PE (n = 5/group) women for activation and expression of cytotoxic markers using flow cytometry to investigate a possible correlation with placental expression of ERAP1/2. Finally, we characterized the cytokines released from immune cells isolated from normotensive women and those with PE, stimulated ex vivo by JEG-3 trophoblast cells. The ERAP1 protein was significantly upregulated in first trimester placentae compared to placentae at delivery from both normotensive and PE women (p < 0.05): expression of placental ERAP1 protein was also relatively higher in normotensive than PE women. Although the protein expression of both ERAP1/ERAP2 was significantly lower in women with PE compared to normotensive controls (p < 0.05), ERAP2 protein expression remained unchanged in normotensive women at delivery compared to expression in the first trimester. Flow cytometry analysis revealed an increase in activation and cytotoxic natural killer (NK) cells in peripheral blood of PE compared to normotensive women. Intriguingly, there was a notable difference in cytokine release from the activated immune cells when further stimulated by trophoblast cells. The immune cells from PE released elevated expressions of interleukin (IL)-2, IL-4, and most notably, pro-inflammatory IL-13 and IL-17α, inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to normal peripheral blood mononuclear cells (PBMCs). Taken together, these findings suggest that differential lymphocyte activation could be associated with altered ERAP1/ERAP2 expression.

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real-time polymerase chain reaction (qRT-PCR), promoter DNA methylation, qRT-PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG-3 (trophoblast), and HMEC-1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor-2 and fatty acid-binding protein-4 (FABP4) in these cells. Curcumin-stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA-G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down-regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA-G by curcumin, hitherto unknown, is a novel finding since HLA-G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.

RETRACTED ARTICLE: Effect of Maternal Iron Deficiency Anaemia on the Expression of Iron Transport Proteins in the Third Trimester Placenta

We, the Editors and Publisher of the journal Fetal and Pediatric Pathology, have retracted the following article: Mullapudi Venkata Surekha, Thathapudi Sujatha, Shravanthi Gadhiraju, Sandeep Kumar Kotturu, Mudili Siva Prasad, K. Sarada, Varanasi Bhaskar & Putcha Uday Kumar (2021) Effect of Maternal Iron Deficiency Anaemia on the Expression of Iron Transport Proteins in the Third Trimester Placenta, Fetal and Pediatric Pathology, 40:6, 581-596, DOI: https://doi.org/10.1080/15513815.2020.1725942 After publication of this article, concerns were raised to the Editor and Publisher regarding the duplication of a number of figures across the above and following manuscripts: M. V. Surekha, Thathapudi Sujatha, Shravanthi Gadhiraju, Putcha Uday Kumar, Sandeep Kumar Kotturu, Krishnakumar Sharada & V. Bhaskar (2022) Impact of maternal iron deficiency anaemia on the expression of the newly discovered multi-copper ferroxidase, Zyklopen, in term placentas, Journal of Obstetrics and Gynaecology, 42:1, 74-82, DOI: 10.1080/01443615.2021.1882968 Mullapudi Venkata Surekha, Thathapudi Sujatha, Shravanthi Gadhiraju, Putcha Uday Kumar, Mudili Siva Prasad, Gummadi Sailaja, V. Bhaskar & Thimmapuram Srinivas (2022) Expression of iron transport protein Divalent metal transporter 1 (DMT1) increases in response to maternal iron deficiency anemia in near term to term placenta, The Journal of Maternal-Fetal & Neonatal Medicine, 35:6, 1045-1053, DOI: 10.1080/14767058.2020.1742317 The authors were contacted and, while they cooperated with the investigation and confirmed there were errors present in the article, they were unable to address the concerns raised. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. The corresponding author listed in this publication has been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Characteristics of Natural Killer Cell Interaction with Trophoblast Cells During Pregnancy.

Maternal natural killer cells (NK cells) are a prevailing leukocyte population in the uteroplacental bed. Current descriptions of the effect of cytokines from the placental microenvironment on the expression of receptors by trophoblast and NK cells are inadequate and contradictory. There is insufficient information about the ability of NK cells to migrate through trophoblast cells. To assess the impact of conditioned media obtained during culturing of placentas from the first and the third trimesters of healthy pregnancies on the phenotype of trophoblast and NK cells and impact on adhesion and transmigration of NK cells through trophoblast cell layer. We established that conditioned media obtained from both first and third trimester placentas increased the intensity of CD106, CD49e, CD49a, CD31, CD51/61, and integrin β6 expression by trophoblast cells. Conditioned media obtained from first trimester placentas increased the intensity of CD11a, CD29, CD49d, CD58, CD29 expression by NK cells. The presence of conditioned media from third trimester placentas resulted in more intense CD29, CD49d, CD11a, CD29, CD49d, and CD58 expression by NK cells. Migration of NK cells through trophoblast cells in the presence of conditioned media from first trimester placentas was increased compared with the migration level in the presence of conditioned media from third trimester placentas. This may be associated with increased expression of CD18 by NK cells. First trimester placental secretory products increase adhesion receptor expression by both trophoblast and NK cells. Under these conditions, trophoblast is capable of ensuring NK cell adhesion and transmigration.

140: Placenta accreta index predicts placenta accreta spectrum severe enough to result in hysterectomy

To validate the Placenta Accreta Index (PAI) for prenatal diagnosis of placenta accreta spectrum (PAS) at time of delivery in women with prior cesarean deliveries. Using an ultrasound (US) database, pregnancies complicated by third trimester placenta previa or low-lying placenta in the setting of prior cesarean delivery (CD) between 1/2012 and 12/2018 were identified. Images from the third trimester US most immediately preceding delivery were read by two physicians who were blinded to US reports, pregnancy outcomes, and clinical outcomes. A PAI calculation was calculated. This calculation includes number of prior cesareans, placental location, lacunae, bridging vessels, and smallest myometrial thickness. PAS was diagnosed based on histology of degree of placenta invasion (accreta, increta, or percreta). Clinical outcomes were obtained. Statistical analysis included Wilcoxon rank-sum test with P < 0.05 considered significant. 194 pregnancies met inclusion criteria. Cesarean hysterectomy was performed in 93 (48%), repeat CD in 94 (48%), and vaginal delivery in 7 (4%). Of those who underwent hysterectomy, 79 had histologic confirmation of PAS. The median PAI score was significantly different according to need for hysterectomy with the diagnosis of PAS (p < 0.001). Of the remaining 14 who underwent hysterectomy, but did not have histologic confirmation of PAS, 12 had clinically suspected invasion and 11 had bleeding complications. The PAI is useful in the prenatal diagnosis of PAS at the time of delivery and provides a standardized approach to US diagnosis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Expression of Retinoid Acid Receptor-Responsive Genes in Rodent Models of Placental Pathology.

In humans, retinoic acid receptor responders (RARRES) have been shown to be altered in third trimester placentas complicated by the pathologies preeclampsia (PE) and PE with intrauterine growth restriction (IUGR). Currently, little is known about the role of placental Rarres in rodents. Therefore, we examined the localization and expression of Rarres1 and 2 in placentas obtained from a Wistar rat model of isocaloric maternal protein restriction (E18.5, IUGR-like features) and from an eNOS-knockout mouse model (E15 and E18.5, PE-like features). In both rodent models, Rarres1 and 2 were mainly localized in the placental spongiotrophoblast and giant cells. Their placental expression, as well as the expression of the Rarres2 receptor chemokine-like receptor 1 (CmklR1), was largely unaltered at the examined gestational ages in both animal models. Our results have shown that RARRES1 and 2 may have different expression and roles in human and rodent placentas, thereby underlining immanent limitations of comparative interspecies placentology. Further functional studies are required to elucidate the potential involvement of these proteins in early placentogenesis.

Classics revisited: Anna Reinstein-Mogilowa's observations on uterine glands and the cytotrophoblastic shell in the first trimester of human pregnancy

BackgroundAnna Reinstein-Mogilowa was the first woman to publish a journal article on placenta. She was among several women from Imperial Russia to study medicine at Swiss universities in the late nineteenth century. FindingsHer observations on first trimester placenta built on those of her supervisor Theodor Langhans and a study of term placenta by her compatriot Raissa Nitabuch. She established the fetal origin of what is now known as the cytotrophoblastic shell. In addition, she made a close study of the uterine glands concluding that they did not connect to the intervillous space. Her subsequent career as an obstetrician was in a suburb of Buffalo, New York. Together with her husband and daughter she was a political activist. DiscussionAnna Reinstein-Mogilowa's life is discussed in the context of contemporary women in science including Raissa Nitabuch and Eva Chaletzy/Haljecka. Their stories are interpreted against the historical background of obstacles to the study and practise of medicine faced by nineteenth century women.

Reduced hepatic metallothionein expression in first trimester fetuses in response to intrauterine smoking exposure: a consequence of low maternal zinc levels?

Does maternal smoking in early pregnancy affect metallothionein 1 and 2 (MT1 and MT2) mRNA and protein expression in first trimester placenta or embryonic/fetal liver? In the first trimester, MT protein expression is seen only in liver, where smoking is associated with a significantly reduced expression. Zinc homeostasis is altered by smoking. Smoking induces MT in the blood of smokers properly as a result of the cadmium binding capacities of MT. In term placenta MT is present and smoking induces gene and protein expression (MT2 in particular), but the MT presence and response to smoking have never been examined in first trimester placenta or embryonic/fetal tissues. Cross sectional study where the presence of MT mRNA and protein was examined at the time of the abortion. The material was collected with informed consent after surgical intervention and frozen immediately. For protein expression analysis, liver tissue originating from smoking exposed n = 10 and unexposed n = 12 pregnancies was used. For mRNA expression analyses, placental tissue originating from smokers n = 19 and non-smokers n = 23 and fetal liver tissue from smoking exposed n = 16 and smoking unexposed pregnancies n = 13, respectively, were used. Tissues were obtained from women who voluntarily and legally chose to terminate their pregnancy between gestational week 6 and 12. Western blot was used to determine the protein expression of MT, and real-time PCR was used to quantify the mRNA expression of MT2A and eight MT1 genes alongside the expression of key placental zinc transporters: zinc transporter protein-1 (ZNT1), Zrt-, Irt-related protein-8 and -14 (ZIP8 and ZIP14). A significant reduction in the protein expression of MT1/2 in liver tissue (P = 0.023) was found by western blot using antibodies detecting both MT forms. Overall, a similar tendency was observed on the mRNA level although not statistically significant. Protein expression was not present in placenta, but the mRNA regulation suggested a down regulation of MT as well. A suggested mechanism based on the known role of MT in zinc homeostasis could be that the findings reflect reduced levels of easily accessible zinc in the blood of pregnant smokers and hence a reduced MT response in smoking exposed fetal/embryonic tissues. Smoking was based on self-reports; however, our previous studies have shown high consistency regarding cotinine residues and smoking status. Passive smoking could interfere but was found mainly among smokers. The number of fetuses was limited, and other factors such as medication and alcohol might affect the findings. Information on alcohol was not consistently obtained, and we cannot exclude that it was more readily obtained from non-users. In the study, alcohol consumption was reported by a limited number (less than 1 out of 5) of women but with more smokers consuming alcohol. However, the alcohol consumption reported was typically limited to one or few times low doses. The interaction between alcohol and smoking is discussed in the paper. Notably we would have liked to measure zinc status to test our hypothesis, but maternal blood samples were not available. Zinc deficiency-in particular severe zinc deficiency-can affect pregnancy outcome and growth. Our findings indicate that zinc homeostasis is also affected in early pregnancy of smokers, and we know from pilot studies that even among women who want to keep their babies, the zinc status is low. Our findings support that zinc supplements should be considered in particular to women who smoke. We thank the Department of Biomedicine for providing laboratory facilities and laboratory technicians and the Lundbeck Foundation and Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis Legat for financial support. The authors have no competing interests to declare. N/A.

Dysregulation of Placental Functions and Immune Pathways in Complete Hydatidiform Moles.

Gene expression studies of molar pregnancy have been limited to a small number of candidate loci. We analyzed high-dimensional RNA and protein data to characterize molecular features of complete hydatidiform moles (CHMs) and corresponding pathologic pathways. CHMs and first trimester placentas were collected, histopathologically examined, then flash-frozen or paraffin-embedded. Frozen CHMs and control placentas were subjected to RNA-Seq, with resulting data and published placental RNA-Seq data subjected to bioinformatics analyses. Paraffin-embedded tissues from CHMs and control placentas were used for tissue microarray (TMA) construction, immunohistochemistry, and immunoscoring for galectin-14. Of the 14,022 protein-coding genes expressed in all samples, 3,729 were differentially expressed (DE) in CHMs, of which 72% were up-regulated. DE genes were enriched in placenta-specific genes (OR = 1.88, p = 0.0001), of which 79% were down-regulated, imprinted genes (OR = 2.38, p = 1.54 × 10−6), and immune genes (OR = 1.82, p = 7.34 × 10−18), of which 73% were up-regulated. DNA methylation-related enzymes and histone demethylases were dysregulated. “Cytokine–cytokine receptor interaction” was the most impacted of 38 dysregulated pathways, among which 17 were immune-related pathways. TMA-based immunoscoring validated the lower expression of galectin-14 in CHM. In conclusion, placental functions were down-regulated, imprinted gene expression was altered, and immune pathways were activated, indicating complex dysregulation of placental developmental and immune processes in CHMs.

Transcriptomic features of trophoblast lineage cells derived from human induced pluripotent stem cells treated with BMP 4

IntroductionEarly development of the human placenta remains poorly understood due to the lack of proper model systems. Previous reports have demonstrated that human induced pluripotent stem cells (hiPSCs) treated with bone morphogenetic protein 4 (BMP4) can differentiate into extraembryonic tissues as useful models of the early stage of trophoblast (TB) differentiation. In our previous study, we optimized the culture conditions of hiPSC-derived TB lineages, but the differentiated cells were heterogeneous. MethodsIn order to characterize the hiPSC-derived TB lineage cells, four types of hiPSCs were treated with 50 ng/mL of BMP4 for 10 days. Subsequently, cells that were positive for the pan-TB marker keratin 7(KRT7) were purified from the differentiated cells using flow cytometry and identified with a DNA microarray. ResultsComparisons of our microarray data with the human transcriptome in a previous large-scale analysis showed that the gene expression patterns of KRT7+ cells were similar to the placenta. In total, 259 upregulated genes were commonly expressed in all four KRT7+ groups, including well-known TB markers. Among these upregulated genes, several with poorly investigated expression patterns and functions were confirmed as expressed in the primary placenta. While only XAGE2 and KCNQ2 were expressed in TB layers, XAGE2 was expressed throughout pregnancy and KCNQ2 was expressed only in cytotrophoblasts of the first trimester placenta. ConclusionBMP4-treated KRT7+ cells were in the course of the human placental development. In addition, this approach allowed the identification of new genes that might be involved in placentation. However, further studies are needed to confirm their functions.

Tissue stiffness at the human maternal-fetal interface.

STUDY QUESTIONWhat is the stiffness (elastic modulus) of human nonpregnant secretory phase endometrium, first trimester decidua, and placenta?SUMMARY ANSWERThe stiffness of decidua basalis, the site of placental invasion, was an order of magnitude higher at 103 Pa compared to 102 Pa for decidua parietalis, nonpregnant endometrium and placenta.WHAT IS KNOWN ALREADYMechanical forces have profound effects on cell behavior, regulating both cell differentiation and migration. Despite their importance, very little is known about their effects on blastocyst implantation and trophoblast migration during placental development because of the lack of mechanical characterization at the human maternal–fetal interface.STUDY DESIGN, SIZE, DURATIONAn observational study was conducted to measure the stiffness of ex vivo samples of human nonpregnant secretory endometrium (N = 5) and first trimester decidua basalis (N = 6), decidua parietalis (N = 5), and placenta (N = 5). The stiffness of the artificial extracellular matrix (ECM), Matrigel®, commonly used to study migration of extravillous trophoblast (EVT) in three dimensions and to culture endometrial and placental organoids, was also determined (N = 5).PARTICIPANTS/MATERIALS, SETTING, METHODSAtomic force microscopy was used to perform ex vivo direct measurements to determine the stiffness of fresh tissue samples. Decidua was stained by immunohistochemistry (IHC) for HLA-G+ EVT to confirm whether samples were decidua basalis or decidua parietalis. Endometrium was stained with hematoxylin and eosin to confirm the presence of luminal epithelium. Single-cell RNA sequencing data were analyzed to determine expression of ECM transcripts by decidual and placental cells. Fibrillin 1, a protein identified by these data, was stained by IHC in decidua basalis.MAIN RESULTS AND THE ROLE OF CHANCEWe observed that decidua basalis was significantly stiffer than decidua parietalis, at 1250 and 171 Pa, respectively (P < 0.05). The stiffness of decidua parietalis was similar to nonpregnant endometrium and placental tissue (250 and 232 Pa, respectively). These findings suggest that it is the presence of invading EVT that is driving the increase in stiffness in decidua basalis. The stiffness of Matrigel® was found to be 331 Pa, significantly lower than decidua basalis (P < 0.05).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONTissue stiffness was derived by ex vivo measurements on blocks of fresh tissue in the absence of blood flow. The nonpregnant endometrium samples were obtained from women undergoing treatment for infertility. These may not reflect the stiffness of endometrium from normal fertile women.WIDER IMPLICATIONS OF THE FINDINGSThese results provide direct measurements of tissue stiffness during the window of implantation and first trimester of human pregnancy. They serve as a basis of future studies exploring the impact of mechanics on embryo implantation and development of the placenta. The findings provide important baseline data to inform matrix stiffness requirements when developing in vitro models of trophoblast stem cell development and migration that more closely resemble the decidua in vivo.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the Centre for Trophoblast Research, the Wellcome Trust (090108/Z/09/Z, 085992/Z/08/Z), the Medical Research Council (MR/P001092/1), the European Research Council (772426), an Engineering and Physical Sciences Research Council Doctoral Training Award (1354760), a UK Medical Research Council and Sackler Foundation Doctoral Training Grant (RG70550) and a Wellcome Trust Doctoral Studentship (215226/Z/19/Z).

Glycoanalysis of the placental membrane glycoproteins throughout placental development

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.