Placental Response Research Articles

Severe SARS-CoV-2 placenta infection can impact neonatal outcome in the absence of vertical transmission.

The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the pathophysiology of the placenta and its impact on pregnancy outcome has not yet been fully elucidated. Here, we present a comprehensive clinical, morphological, and molecular analysis of placental tissues from pregnant women with and without SARS-CoV-2 infection. SARS-CoV-2 could be detected in half of placental tissues from SARS-CoV-2-positive women. The presence of the virus was not associated with any distinctive pathological, maternal, or neonatal outcome features. SARS-CoV-2 tissue load was low in all but one patient who exhibited severe placental damage leading to neonatal neurological manifestations. The placental transcriptional response induced by high viral load of SARS-CoV-2 showed an immunopathology phenotype similar to autopsy lung tissues from patients with severe coronavirus disease 2019. This finding contrasted with the lack of inflammatory response in placental tissues from SARS-CoV-2-positive women with low viral tissue load and from SARS-CoV-2-negative women. Importantly, no evidence of vertical transmission of SARS-CoV-2 was found in any newborns, suggesting that the placenta may be an effective maternal-neonatal barrier against the virus even in the presence of severe infection. Our observations suggest that severe placental damage induced by the virus may be detrimental for the neonate independently of vertical transmission.

Placental Immune Responses to Viruses: Molecular and Histo-Pathologic Perspectives.

As most recently demonstrated by the SARS-CoV-2 pandemic, congenital and perinatal infections are of significant concern to the pregnant population as compared to the general population. These outcomes can range from no apparent impact all the way to spontaneous abortion or fetal infection with long term developmental consequences. While some pathogens have developed mechanisms to cross the placenta and directly infect the fetus, other pathogens lead to an upregulation in maternal or placental inflammation that can indirectly cause harm. The placenta is a temporary, yet critical organ that serves multiple important functions during gestation including facilitation of fetal nutrition, oxygenation, and prevention of fetal infection in utero. Here, we review trophoblast cell immunology and the molecular mechanisms utilized to protect the fetus from infection. Lastly, we discuss consequences in the placenta when these protections fail and the histopathologic result following infection.

Effects of ‘rescue’ dose of antenatal corticosteroids on placental histopathology in preterm births

IntroductionAntenatal corticosteroids (ACS) are frequently used to reduce neonatal morbidity in preterm births (PTBs). A ‘rescue’ dose of ACS can be administer, if the risk of PTB remains. Some reports indicated that repeated doses of ACS might impact placental histology and possibly its function. We aimed to study whether repeated doses of ACS effect placental histopathology and pregnancy outcome. MethodsThe medical files and placental reports of all PTB, at 24-336/7 weeks, between Nov 2008–Dec 2019, were reviewed. The study population was divided into three groups; no-ACS (PTBs without ACS treatment), one-ACS (PTBs after a full or partial ACS course), and rescue-ACS (PTBs after a ‘rescue’ course of ACS). Placental lesions were classified according to “Amsterdam” criteria into maternal and fetal vascular malperfusion lesions, maternal and fetal inflammatory responses and chronic villitis. Placental lesions and pregnancy outcome were compared between the study groups. ResultsThe no-ACS group (n = 58) was characterized by increased rates of PTB<28 weeks (p = 0.003), perinatal death (p < 0.001) and composite neonatal infectious morbidity (p = 0.022), as compared to the one-ACS group (n = 331) and the rescue-ACS group (n = 53). Placental MIR lesions were more common among the rescue-ACS group, compared to the one- and no-ACS groups (p = 0.022). Other placental lesions did not differ between the groups. On multivariate logistic regression analysis, MIR lesions were independently associated with rescue-ACS treatment (aOR 3.00, 95% CI 1.10–8/17, p = 0.031). DiscussionRescue course of ACS is associated with increased rate of placental maternal inflammatory response. These findings probably result from maternal stress stimuli without an adverse impact on early neonatal outcome.

Preconceptional Lipid-Based Nutrient Supplementation in 2 Low-Resource Countries Results in Distinctly Different IGF-1/mTOR Placental Responses

BackgroundPreconceptional maternal small-quantity lipid-based nutrient supplementation (SQLNS) improved intrauterine linear growth in low-resource countries as demonstrated by the Women First Preconception Maternal Nutrition Trial (WF). Fetal growth is dependent on nutrient availability and regulated by insulin-like growth factor 1 (IGF-1) through changes in placental transfer capacity, mediated by the mechanistic target of rapamycin (mTOR) pathway. ObjectivesOur objective was to evaluate the role of placental mTOR and IGF-1 signaling on fetal growth in women from 2 low-resource countries with high rates of stunting after they received preconceptional SQLNS. MethodsWe studied 48 women from preconception through delivery who were from Guatemala and Pakistan and received SQLNS or not, as part of the WF study. Placental samples were obtained at delivery (control, n = 24; SQLNS, n = 24). Placental protein or mRNA expression of eukaryotic translation initiation factor binding protein-1 (4E-BP1), ribosomal protein S6 (rpS6), AMP-activated protein kinase α (AMPKA), IGF-1, insulin-like growth factor receptor (IGF-1R), and pregnancy associated plasma protein (PAPP)-A, and DNA methylation of the IGF1 promoter were determined. Maternal serum IGF-1, insulin-like growth factor binding protein (IGFBP)-3, IGFBP-4, IGFBP-5, PAPP-A, PAPP-A2, and zinc were measured. ResultsMean ± SEM maternal prepregnancy BMI differed between participants in Guatemala (26.5 ± 1.3) and Pakistan (19.8 ± 0.7) (P < 0.001). In Pakistani participants, SQLNS increased the placental rpS6(T37/46):rpS6 ratio (1.5-fold) and decreased the AMPKA(T172):AMPKA ratio. Placental IGF1 mRNA expression was positively correlated with birth length and birth weight z-scores. Placental PAPP-A (30-fold) and maternal serum zinc (1.2-fold) increased with SQLNS. In Guatemalan participants SQLNS did not influence placental mTOR signaling. Placental IGF-1R protein expression was positively associated with birth length and birth weight z-scores. SQLNS increased placental PAPP-A (40-fold) and maternal serum IGFBP-4 (1.6-fold). ConclusionsIn Pakistani pregnant women with poor nutritional status, preconceptional SQLNS activated placental mTOR and IGF-1 signaling and was associated with improved fetal growth. In contrast, in Guatemalan women SQLNS did not activate placental nutrient-sensing pathways. In populations experiencing childhood stunting, preconceptional SQLNS improves placental function and fetal growth only in the context of poor maternal nutrition. This trial was registered at clinicaltrials.gov as NCT01883193.

Interleukin-6 pathobiology in equine placental infection.

Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. Sub-acute ascending placentitis was induced via trans-cervical inoculation of Szooepidemicus, and various fluids/serum/tissues collected 8days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n=6) and control (n=6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

Exposure to Gestational Diabetes Mellitus (GDM) alters DNA methylation in placenta and fetal cord blood

BackgroundFetal exposure to maternal GDM increases offspring risk for adult-onset metabolic syndromes. Epigenetic modifications such as DNA methylation are modulators for fetal metabolic programming and susceptibility to adult-onset disease. This study investigates genome-wide DNA methylation in GDM exposed cord blood and placenta. MethodsOral glucose tolerance testing between 24 and 28 weeks of pregnancy was used to determine severity of glucose intolerance. We measured DNA methylation (DNAm) using the Illumina Infinium 450 K array in 42 fetal cord blood and 36 placenta samples. ResultsWe identified 662 and 99 CpG sites in GDM placenta and cord blood, respectively at p-value <0.01 and a methylation difference >5% after adjustment for confounders. Annotated sites for AHRR and PTPRN2 were common to cord blood and placenta. Adding published GDM cord blood DNAm data we segregated patients based on treatment (Diet Only vs. +Insulin) and identified altered CpG sites to be unique to each GDM treatment group. ConclusionConsistency in findings with other studies provides evidence for the role of DNAm in placental and fetal responses to hyperglycemia. However, segregating DNAm analysis of GDM samples based on treatment may help delineate whether observed DNAm alterations are reflective of adaptive responses or treatment effects in utero.

Heat stress affects the expression of key genes in the placenta, placental characteristics, and efficiency of Saanen goats and the survival and growth of their kids

Heat stress is detrimental during gestation; however, the effects of heat stress on goat placental characteristics and kid survival remain unclear. The objective of this study was to evaluate the effects of heat stress at final gestation on cortisol concentration, placenta characteristics, and the expression of genes related to placenta. Forty-six primiparous and multiparous Saanen goats were subjected to control (CT; under a thermoneutral environment: air temperature between 12°C and 25°C and the relative humidity from 45 to 73%, n = 23) or heat stress (HS; under a climatic chamber: air temperature at 37°C and the relative humidity at 60 to 70% from 0800 to 1600 h, n = 23) from the last 60 d of pregnancy until the first colostrum suckling. The heat challenge imposed on HS goats during the prepartum period increased their rectal temperature, respiratory frequency, and cortisol levels in plasma and amniotic fluid versus CT goats. In the placenta, HS treatment also increased the expression of the HSPA1A gene. Heat-stressed goats also showed significantly lower expression of HSD11B2 and greater expression of MC2R and NR3C1 than CT goats, suggesting that heat stress decreased the effectiveness by which the HSD11B2 enzyme converts cortisol to cortisone and increased placental responsiveness to cortisol. The HS goats took longer to release the placenta with lighter placental cotyledons, and HS goats had a lower ratio between the kid's weight at birth and placenta weight than CT goats. There was no treatment effect on the kids' survival or weights at birth, but the kids from goats subjected to HS presented lesser cortisol concentration and greater mortality rates at weaning than kids from CT goats. Finally, the overexpression of HSPA1A by HS goats suggests a protective response of placenta. However, the heat stress negatively affected the placenta's expulsion length, placental cotyledons number, weight and area, the ratio between kid's weight and placenta weight, and cortisol signaling. Indeed, the upregulation of MC2R and NR3C1 and downregulation of HSD11B2 on placenta caused by heat stress were associated with greater cortisol concentrations in the amniotic fluid of HS goats. Although HS and CT kids had adequate weights and survival rate during the first weeks of life, the heat stress increased the mortality at weaning of HS kids versus CT kids, suggesting that the heat stress effect persists and can change the ability of kids to respond to weaning challenge.

77 Role of Placental PAPP-A and IGF-II in placental growth in hypoxia-induced fetal growth restriction

Fetal growth restriction (FGR) is associated with an increased risk of perinatal and neonatal mortality. Insulin-like growth factors (IGFs) play a major role in fetal growth through PAPP-A-IGF signaling. IGF-II, specifically, is involved in development of placental exchange into late gestation. Chronic hypoxia can result in FGR through mechanisms of placental dysfunction. We hypothesize that chronic hypoxia-exposure decreases placental PAPPA- and IGF-II protein expression resulting in FGR. Female guinea pigs (GPs) were time-mated with male GPs. Pregnant sows (term gestation = 65 days) were randomly assigned to either normoxia (NMX, room air, 21% O2) or hypoxia (HPX, 10.5% O2) during the last 14 days of pregnancy. At near-term gestation, anesthetized female fetuses were removed via hysterotomy. Fetal body, organ (brain, liver, heart) and placenta weights (wt) were recorded. Asymmetric FGR was classified as fetal wt 0.66. Placental tissues of 7 NMX and 7 HPX-FGR male GPs were homogenized and protein expression of PAPP-A and IGF-II was measured by Western blot. NMX and HPX were compared using Student’s t-Tests with statistical significance determined at p value < 0.05. In the NMX and HPX-FGR cohorts, there was no statistical difference in the mean gestational age at delivery 63.7d and 62.4d (p=0.07), respectively. As expected, fetal body wt, brain, and liver wt were significantly less in the HPX-FGR cohort as compared to NMX (Table 1). Relative placenta wt was increased by 25% with HPX (p<0.05). There was no statistical difference in PAPP-A expression in NMX and HPX placentas (1.22 ± .28 vs 1.55± .79, p=0.353, respectively). IGF-II expression was significantly increased in HPX-FGR placentas (0.58 ± .13 vs 1.22 ±.34, p=0.05), (Figure 1). Contrary to our hypothesis, neither PAPP-A nor IGF-II were decreased with hypoxia. Rather, hypoxia increased IGF-II, which is consistent with an increase in relative placental wt. This suggests a unique role for IGF-II as mechanism of compensatory placental growth response, despite the presence of FGR.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

93 Characterizing the key mediators of SARS-CoV-2 infection in the human placenta: is vertical transmission possible?

Previous coronavirus outbreaks (SARS and MERS) were linked to severe pregnancy outcomes including miscarriage and stillbirth. With exception of anecdotal reports, vertical transmission of SARS-CoV2 remains unproven. Emerging evidence has implicated the angiotensin converting enzyme 2 (ACE2) in SARS-CoV-2 viral entry and its pro-inflammatory effects with respiratory syndromes. The serine protease TMPRSS2 is required for spike protein priming and subsequent viral replication. Little is understood regarding the expression of ACE2 and TMPRSS2 in human placentas. We examined these proteins in term human placentas and maternal blood. Unlabored, term cesarean placentas were collected from uncomplicated pregnancies (n=10). Gene expression and western blot analyses for ACE2 and TMPRSS2 was conducted using intervillous biopsies. Placenta proteins were compared to commercially available human lung samples, a human cytotrophoblasts (BeWo cells) and recombinant proteins. Previously banked maternal plasma samples from uncomplicated pregnancies across gestation were analyzed by ELISA (n =12 donors). Placental ACE2 expression was significantly greater than TMPRSS2 (Figure1.A). Both were present at predicted sizes corresponding to recombinant proteins and in greater amount than lung samples. Qualitatively, the abundance of ACE2 in placenta samples corresponds with 1ng of recombinant protein vs. 0.1ng for TMPRSS2 (Figure1.B). BeWo cells appear to have a greater amount of TMPRSS2 vs. ACE2. In maternal plasma, ACE2 was highly variable and undetectable most samples (Figure 2). Despite the few reports of vertical transmission, our results indicate human placentas have endogenous ACE2 and TMPRSS2 and possibly at greater levels than the lung. ELISA results suggest that maternal blood is not a major contributor of placental ACE2 in most pregnancies. Since the proteins necessary for SARS-CoV-2 infection are present, other element(s) may influence or are required for vertical transmission. Future studies planned to address placental innate immune responses against this variant of coronavirus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

288 Assessing in vivo placental function in pregnancies complicated by maternal diabetes mellitus

We sought to examine fetal brain and placental response to maternal hyperoxia using blood oxygen level–dependent (BOLD) magnetic resonance imaging (MRI) in pregnancies complicated by diabetes mellitus (DM). This is a single center prospective case control cohort study of women with singleton gestations, at least 18 weeks or greater. Cases include women diagnosed with either pre-gestational or gestational DM. Controls include healthy pregnancies without any comorbidities. Gradient-echo planar and fast spin-echo T2 weighted imaging was performed with a maternal hyperoxia paradigm of 2 minutes resting state baseline (RS), 6 minutes maternal hyperoxia (HO) of 100% oxygen via a non-rebreather and 5.6 minutes post oxygenation for return to resting state (RR). Analyses were performed at one minute time intervals to assess the change in R2* from baseline (ΔR2*). Student’s t-test was performed to compare outcomes between groups. Of the 113 women recruited, 67 (59.3%) healthy controls with mean gestational age (GA) in weeks 30.9 ± 4.3, and 46 (40.7%) cases, mean GA 30.2 ± 4.5. Of the cases 13 (28.26%) had Type 1 DM, 15 (32.61%) were diagnosed with Type 2 DM, 17 (36.96%) had Gestational (G) DM and 1 (2.17%) subject never disclosed the diabetes diagnosis. Overall, results were similar between groups during HO. During RR minutes 4 and 5, the ΔR2 (difference in oxygen signal relative to baseline), was lower in the diabetic cohort compared to controls, with most significant decrease in T1DM (RR6) and T2DM (RR2, RR4) (Fig 1&2). Metabolic, inflammatory and vascular changes have been described in ex vivo placentas from diabetic pregnancies. Maternal hyperoxia during BOLD imaging reflects oxygen carrying capacity and transfer from the maternal circulation to the placenta and fetus. Decreased BOLD signaling suggests altered placenta function, reflecting either increased oxygen consumption of the diabetic placenta or continued oxygen transfer to the fetus. Additional studies to explore the clinical significance of these findings are underway.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Placental Expression and Relative Role of Anti-inflammatory Annexin A1 and Animal Lectin Galectin-3 in the Pathogenesis of Preeclampsia.

Preeclampsia (PE) remains the major cause for maternal and foetal mortality and morbidity all over the world. Preeclampsia is associated with maternal, placental aggravated inflammatory response and generalized endothelial damage. AnnexinA1 (AnxA1) is glucocorticoid regulated protein regulates a wide range of cellular and molecular steps of the inflammatory response and is implicated in resolution of inflammation. Galectin-3 (Gal-3), β-galcotoside-binding lectin participates in many functions, both intra- and extracellularly. Recently it has been shown that galectin-3 modulates the inflammation. Role of AnxA1 and Galectin-3 is poorly studied in context with human reproductive disease like Preeclampsia. Therefore, the present study examined the expression of AnxA1 and Gal-3 which are involved in modulation of inflammation and their association in the placental bed of pregnancy with and without PE. The study group consisted of placental bed biopsy tissues obtained from pregnancies with PE (n = 30) and without (n = 30) PE. The expression of AnxA1 and Gal-3 in the placental bed tissues was evaluated quantitatively using Immunohisto-chemistry (IHC), western blot and mRNA expression analysis by quantitative RT-PCR. Our IHC, western blot and RT PCR analyses showed the increase in the expression of AnxA1 and Gal-3 in PE group compared with the normotensive control group (P < 0.001). The increased expression of AnxA1 and Gal-3 in placental bed may be associated with a systemic inflammatory response in PE, suggesting role of AnxA1 and Gal-3 in PE pathogenesis.

Impact of placental malaria on maternal, placental and fetal cord responses and its role in pregnancy outcomes in women from Blue Nile State, Sudan

BackgroundThe sequestration of Plasmodium falciparum infected cells in the placenta results in placental malaria (PM). It activates the mother's immune cells and induces secretion of inflammatory cytokines, which might influence pregnancy outcomes. This study aims to investigate the cytokines (levels IL-4, IL-6, IL-10, IL-17A, and INF γ) in maternal peripheral, placental, and umbilical cord blood in response to PM and the extent to which this may influence maternal haemoglobin levels and birth weight.MethodsA total of 185 consenting Sudanese women from Blue Nile State were enrolled at delivery time in a cross-sectional study conducted between Jan 2012-Dec 2015. Malaria infection in the collected maternal peripheral, placental, umbilical cord samples was determined microscopically, and ELISA was used to measure the plasma levels IL-4, IL-6, IL-10, IL-17A, and INF γ in the collected positive and negative malaria samples.ResultsElevated levels of IL-4 and IL-10 and reduced levels of IL-6 were detected in the malaria positive samples in comparison to the negative ones in the three types of the samples investigated. Maternal, IL-4 and IL-10 were significantly higher in the samples collected from the PM infected group compared to the non-infected control (P < 0.001). While the absence of PM was significantly associated with the IL-6 and maternal IFN-γ levels, maternal IL-17A, placental and umbilical cord IFN-γ levels showed no significant difference (P = 0.214, P = 0.065, P = 0.536, respectively) due to infection. Haemoglobin level and birth weight were increased in the group with high levels of IL-6 and IL-17A, but not in the group with IL-4 and IL-10 levels. While significantly negative correlation was found between IFN-γ levels and birth weight for all three types of samples, only maternal peripheral IFN-γ level was significantly positively correlated with maternal haemoglobin (r = 0.171, P = 0.020).ConclusionThese results suggest that PM induces mother’s immune response and impairs her cytokine profile, which might alter maternal haemoglobin levels and the baby's birth weight.

Differential Expression of Rab5 and Rab7 Small GTPase Proteins in Placental Tissues From Pregnancies Affected by Maternal Coronavirus Disease 2019

PurposeThe majority of pregnancies affected by maternal coronavirus disease 2019 (COVID-19) do not result in fetal transmission. However, several studies have identified parenchymal changes in their placental tissues, suggesting a placental response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the maternal–fetal interface. Although many COVID-19 placental studies have focused on the expression of the canonical SARS-CoV-2 entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2, further characterization of subcellular molecules involved in viral trafficking have not yet been investigated in these tissues. Of interest are Rab proteins, a family of small GTPase proteins that direct intracellular transport between different endocytic organelles. Rab5 and Rab7 in particular have previously been implicated in HIV and cytomegalovirus invasion of placental trophoblast cells in vitro; the localization of these molecules has not been fully characterized within the human maternal–fetal interface, however, or within placental tissues from SARS-CoV-2–infected pregnancies. MethodsUsing fluorescent immunohistochemistry, Rab5 and Rab7 placental localization and comparative fluorescence intensity were explored in a cohort of placental tissues from pregnancies affected by maternal COVID-19 disease (COVID, n = 15) compared with contemporary control subjects (Control, n = 10). Fluorescence intensity was quantified by using corrected total cell fluorescence values. FindingsWithin placental villi, Rab5 was consistently localized in syncytiotrophoblast and cytotrophoblast cells. Rab5 had significantly higher mean (SEM) fluorescence intensity in the COVID cohort (Control, 1.96 [0.16]; COVID, 2.62 [0.09]; P = 0.0014). In contrast, although Rab7 was also localized within placental villous syncytiotrophoblast and cytotrophoblast cells, mean (SEM) Rab7 fluorescence intensity was significantly downregulated in COVID vs Control placentas (Control, 35.9 [4.1]; COVID, 20.1 [0.52]; P = 0.0001). ImplicationsThis differential expression of Rab5 and Rab7 suggests that placental endocytic pathways may be altered at the maternal–fetal interface in pregnancies affected by maternal SARS-CoV-2 infection. As key molecules governing intracellular vesicle transport, including viral trafficking, Rab GTPase proteins may be of interest for ongoing studies examining placental responses to COVID-19 in pregnancy.

Risk of Recurrent Pregnancy Loss in the Ukrainian Population Using a Combined Effect of Genetic Variants: A Case-Control Study.

We assessed the predictive ability of a combined genetic variant panel for the risk of recurrent pregnancy loss (RPL) through a case-control study. Our study sample was from Ukraine and included 114 cases with idiopathic RPL and 106 controls without any pregnancy losses/complications and with at least one healthy child. We genotyped variants within 12 genetic loci reflecting the main biological pathways involved in pregnancy maintenance: blood coagulation (F2, F5, F7, GP1A), hormonal regulation (ESR1, ADRB2), endometrium and placental function (ENOS, ACE), folate metabolism (MTHFR) and inflammatory response (IL6, IL8, IL10). We showed that a genetic risk score (GRS) calculated from the 12 variants was associated with an increased risk of RPL (odds ratio 1.56, 95% CI: 1.21, 2.04, p = 8.7 × 10−4). The receiver operator characteristic (ROC) analysis resulted in an area under the curve (AUC) of 0.64 (95% CI: 0.57, 0.72), indicating an improved ability of the GRS to classify women with and without RPL. Ιmplementation of the GRS approach can help define women at higher risk of complex multifactorial conditions such as RPL. Future well-powered genome-wide association studies will help in dissecting biological pathways previously unknown for RPL and further improve the identification of women with RPL susceptibility.

Adaptations of the human placenta to hypoxia: opportunities for interventions in fetal growth restriction.

The placenta is the functional interface between the mother and the fetus during pregnancy, and a critical determinant of fetal growth and life-long health. In the first trimester, it develops under a low-oxygen environment, which is essential for the conceptus who has little defense against reactive oxygen species produced during oxidative metabolism. However, failure of invasive trophoblasts to sufficiently remodel uterine arteries toward dilated vessels by the end of the first trimester can lead to reduced/intermittent blood flow, persistent hypoxia and oxidative stress in the placenta with consequences for fetal growth. Fetal growth restriction (FGR) is observed in ∼10% of pregnancies and is frequently seen in association with other pregnancy complications, such as preeclampsia (PE). FGR is one of the main challenges for obstetricians and pediatricians, as smaller fetuses have greater perinatal risks of morbidity and mortality and postnatal risks of neurodevelopmental and cardio-metabolic disorders. The aim of this review was to examine the importance of placental responses to changing oxygen environments during abnormal pregnancy in terms of cellular, molecular and functional changes in order to highlight new therapeutic pathways, and to pinpoint approaches aimed at enhancing oxygen supply and/or mitigating oxidative stress in the placenta as a mean of optimizing fetal growth. An extensive online search of peer-reviewed articles using PubMed was performed with combinations of search terms including pregnancy, placenta, trophoblast, oxygen, hypoxia, high altitude, FGR and PE (last updated in May 2020). Trophoblast differentiation and placental establishment are governed by oxygen availability/hypoxia in early pregnancy. The placental response to late gestational hypoxia includes changes in syncytialization, mitochondrial functions, endoplasmic reticulum stress, hormone production, nutrient handling and angiogenic factor secretion. The nature of these changes depends on the extent of hypoxia, with some responses appearing adaptive and others appearing detrimental to the placental support of fetal growth. Emerging approaches that aim to increase placental oxygen supply and/or reduce the impacts of excessive oxidative stress are promising for their potential to prevent/treat FGR. There are many risks and challenges of intervening during pregnancy that must be considered. The establishment of human trophoblast stem cell lines and organoids will allow further mechanistic studies of the effects of hypoxia and may lead to advanced screening of drugs for use in pregnancies complicated by placental insufficiency/hypoxia. Since no treatments are currently available, a better understanding of placental adaptations to hypoxia would help to develop therapies or repurpose drugs to optimize placental function and fetal growth, with life-long benefits to human health.

Immunomodulatory properties of extracellular vesicles in the dialogue between placental and immune cells.

Extracellular vesicle (EV)-mediated communication has been implicated in the cooperative alliance between trophoblast and immune cells toward maternal tolerance and placentation. Syncytiotrophoblast cells secrete EVs directly into the maternal circulation, which are taken up by immune cells, endothelial cells, and other cell types. Initial evidence also shows that EVs produced by immune cells are, in turn, incorporated by trophoblast cells and modulate placental responses. Non-coding RNAs (ncRNAs), proteins, and lipid mediators transported in EVs are able to influence proliferation, differentiation, cytokine production, and immunological responses of recipient cells. The molecular alphabet and cellular targets involved in this dialogue are being revealed. Nevertheless, several questions regarding the whole content, surface markers, and biological functions of EVs still remain to be investigated in both physiological and pathological conditions. Analysis of circulating EVs in maternal blood has the potential to serve as a minimally invasive approach to monitoring placental functions and immunological features of pregnancy, aiding in the diagnostics of complications. This review addresses the immunomodulatory properties of EVs and their tasks in the communication between placental and immune cells.

Porphyromonas gingivalis Placental Atopobiosis and Inflammatory Responses in Women With Adverse Pregnancy Outcomes.

The microbiome modulates inflammation at the fetal maternal interface on both term and preterm labor. Inflammophilic oral bacteria, such as Porphyromonas gingivalis, as well as urogenital microorganisms (UGM) could translocate to the placenta and activate immune mechanisms in decidual tissue that is associated with adverse pregnancy outcomes (APO). This study establishes the associations between the presence of microbes in the placenta and placental cytokine patterns in women who presented APO, e.g., low birth weight (LBW), preterm premature rupture of membranes (PPROM), preterm birth (PTB) and other clinical signs related to Chorioamnionitis (CA). A total of 40 pregnant women were included in the study and divided into five groups according to placental infection (PI) and APO, as follows: (1) women without PI and without APO (n = 17), (2) women with P. gingivalis-related PI and APO (n = 5), (3) women with P. gingivalis-related PI and without APO (n = 4), (4) women with PI related to UGM and APO (n = 5) and (5) women without PI with APO (n = 9). Obstetric, clinical periodontal status evaluation, and subgingival plaque sampling were performed at the time of delivery. Placental levels of interleukin IL-1β, IL-6, IL-10, IL-15, IL-17A, IL-17F, IL-21, IL-12p70, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 α (MCP-1α), granzyme B, and interferon-γ (IFN-γ) were determined using a multiplex flow cytometry assay. All patients showed a predominant Th-1 cytokine profile related to labor, characterized by IFN-γ overexpression. The analysis by groups suggests that Th-1 profile was trending to maintain cytotoxic cell activity by the expression of IL-15 and granzyme B, except for the group with P. gingivalis-related PI and APO, which exhibited a reduction of IL-10 and IL-17F cytokines (p < 0.05) and a Th-1 profile favoring macrophage activation by MCP-1 production (p < 0.05). This study confirms a pro-inflammatory pattern associated with labor, characterized by a Th-1 profile and the activity of cytotoxic cells, which is enhanced by PI with UGM. However, PI associated with P. gingivalis suggests a switch where the Th-1 profile favors an inflammatory response mediated by MCP-1 and macrophage activity as a mechanistic explanation of its possible relationship with adverse outcomes in pregnancy.

Ex vivo infection of canine and ovine placental explants with Trypanosoma cruzi and Toxoplasma gondii: differential activation of NF kappa B signaling pathways

Chagas disease and toxoplasmosis, caused by Trypanosoma cruzi and Toxoplasma gondii, respectively, are important zoonotic diseases affecting humans, companion animals, and livestock, responsible for major health and economic burden. Both parasites can be transmitted vertically in different mammalian species through the placenta. Of note, the transmission rate of T. cruzi is low in dogs, whereas that of T. gondii is high in sheep. The probability of congenital infection depends on complex parasite-host interactions; parasite factors, maternal and fetal immune responses and placental responses all have a role in infection establishment. Since the innate immune response is regulated, at least partially, by NF-κB signaling pathways, our main objective was to determine the effect of ex vivo infection of canine (CPE) and ovine (OPE) placental explants with both parasites, on the activation of canonical and non-canonical NF-κB pathways and its relation to infection. Here, we show that T. cruzi activates both the NF-κB canonical and non-canonical pathways in CPE and OPE, unlike T. gondii, that activates only the canonical pathway in CPE and has no effect on the non-canonical pathway in both explants. Moreover, the inhibition of either or both NF-κB pathways increases the DNA load of T. cruzi in both explants, modulates, on the other hand, T. gondii infection in a differential fashion. Overall, we conclude that the differential modulation of the NF-κB pathways by both pathogens in placental explants might explain, at least partially, the differences in transmission rates of T. cruzi and T. gondii in different mammalian species.

Iron-Dependent Apoptosis Causes Embryotoxicity in Inflamed and Obese Pregnancy

Since iron is essential for pregnancy, iron supplements are routinely prescribed to pregnant women without screening for iron deficiency. Most women in developed countries have sufficient iron stores, prompting concerns of the potential risks of iron supplementation in iron-replete women. A signal of potential harm of this practice is the association of maternal iron marker ferritin and pregnancy complications. Since ferritin is also induced by inflammation, it is unclear whether iron excess, inflammation, or their interaction cause fetal injury. We used mouse models to address this question. Two mouse models of maternal iron excess were used and compared to mice with normal iron status. To model dietary iron supplementation, C57BL/6 females were fed high-iron diet (2500-5000ppm iron) for 1 week prior to and during pregnancy. To model genetic iron loading, hepcidin knockout females were used and fed standard chow (185ppm iron). Two models of maternal inflammation were used. Acute systemic maternal inflammation was induced by a single subcutaneous injection of LPS on E8.5 or 15.5. Obesity was used as a model of mild chronic systemic maternal inflammation and was induced by feeding female mice Western diet. We evaluated the maternal, placental, and embryo response to iron excess in LPS- and obesity-induced inflammation at various time points. In the model of acute inflammation, we observed an adverse synergistic effect on embryo development when dams were both iron-loaded and LPS-injected, but not with either condition alone. On E8.5, LPS injection in iron-loaded dams caused embryo loss and encephalic malformations (Fig 1a) and on E15.5, LPS injection in iron-loaded dams caused embryo demise and resorption (Fig 1b). Western blotting and immunostaining showed increased apoptotic marker cleaved caspase-3 protein localized to endothelia in placentas and embryos, only when the dam was both iron-loaded and LPS-injected. Cytokine screen in vitro identified TNFα as the signal that synergized with iron to potentiate apoptosis of endothelial cells. Treatment of iron-loaded dams with TNFα-neutralizing antibody protected embryos from LPS-induced death, demonstrating the embryotoxic role of maternal TNFα in iron-loaded dams. Furthermore, RNA-Seq analysis of placental endothelial cells showed enrichment in oxidative stress pathways with maternal iron loading. Treatment of iron-loaded dams with antioxidant α-tocopherol protected against LPS-induced embryo death, attenuated placental inflammation and cleaved caspase-3 expression, and prevented endothelial apoptosis in the placenta and embryo. These data show that iron loading causes endothelial oxidative stress, which sensitizes endothelial cells to inflammation-induced apoptosis. In the obesity model, iron loading worsened embryotoxicity, causing subcutaneous hemorrhaging and eye malformations (Fig 1c), and potentiated placental cleaved caspase-3 expression. Iron loading increased TNFα levels in maternal serum. Importantly, treatment of obese iron-loaded dams with neutralizing TNFα antibody throughout pregnancy reduced the incidence of embryo eye malformations, confirming the role of TNFα in causing embryo injury in iron-loaded obese pregnancy. In summary, maternal iron excess worsened inflammation-induced embryo injury. Iron-dependent embryotoxicity was mediated by TNFα causing lethal apoptotic damage to embryo and placental endothelium which was prevented by antioxidant therapy. Our models raise the possibility that women exposed to both excessive iron and inflammation could be at risk for preventable pregnancy complications. Figure 1. Embryo outcomes in mouse pregnancies with maternal iron loading and inflammation. Figure 1 Disclosures Ganz: Akebia: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company; Global Blood Therapeutics: Consultancy; Ambys: Consultancy; Gossamer Bio: Consultancy; Astellas: Consultancy; Rockwell: Consultancy; Sierra Oncology: Consultancy; Disc Medicine: Consultancy; American Regent: Consultancy. Nemeth:Intrinsic LifeSciences: Current equity holder in private company; Vifor: Consultancy; Protagonist: Consultancy; Silarus Therapeutics: Current equity holder in private company; Ionis Pharmaceuticals: Consultancy.

Differential responses in placenta and fetal thymus at 12\u2009days post infection elucidate mechanisms of viral level and fetal compromise following PRRSV2 infection

BackgroundA pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC).ResultsThe host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways.ConclusionOur data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.