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Dysregulated progenitor cell populations may contribute to poor placental development and placental insufficiency pathogenesis. Side-population cells possess progenitor properties. Recent human trophoblast side-population isolation identified enrichment of 8 specific genes (CXCL8, ELL2, GATA6, HK2, HLA-DPB1, INTS6, SERPINE3 and UPP1) (Gamage et al. 2020, Stem Cell Rev Rep). We characterised these trophoblast side-population markers in human placenta and in placental insufficiency disorders: preeclampsia and fetal growth restriction (FGR). Trophoblast side-population markers localised to mononuclear trophoblasts lining the placental villous basement membrane in preterm control, preeclamptic and FGR placental sections (n = 3, panel of 3 markers/serial section). Analysis of single-cell transcriptomics of an organoid human trophoblast stem cell (hTSC) to extravillous trophoblast (EVT) differentiation model (Shannon et al. 2022, Development) identified that all side-population genes were enriched in mononuclear trophoblast and trophoblasts committed to differentiation under hTSC culture conditions. In vitro validation via 96 h time course hTSC differentiation to EVTs or syncytiotrophoblasts (n = 5) demonstrated ELL2 and HK2 increased with differentiation (p < 0.0024, p < 0.0039 respectively). CXCL8 and HLA-DPB1 were downregulated (p < 0.030, p < 0.011 respectively). GATA6 and INTS6 increased with EVT differentiation only, and UPP1 reduced with syncytialisation. SERPINE3 was undetectable. Trophoblast side-population marker mRNA was measured in human placentas (< 34-weeks' gestation; n = 78 preeclampsia, n = 30 FGR, and n = 18 gestation-matched controls). ELL2, HK2 and CXCL8 were elevated in preeclamptic (p = 0.0006, p < 0.0001, p = 0.0335 respectively) and FGR placentas (p = 0.0065, p < 0.0001, p = 0.0001 respectively) versus controls. Placental GATA6 was reduced in pregnancies with preeclampsia and FGR (p = 0.0014, p = 0.0146 respectively). Placental INTS6 was reduced with FGR only (p < 0.0001). This study identified the localisation of a unique trophoblast subset enriched for side-population markers. Aberrant expression of some side-population markers may indicate disruptions to unique trophoblast subtypes in placental insufficiency. [ABSTRACT FROM AUTHOR]

Spontaneous preterm birth is the leading cause of perinatal morbidity and mortality. Tocolytics are drugs used in cases of imminent preterm birth to inhibit uterine contractions. Nifedipine is a calcium channel blocking agent used to delay threatened spontaneous preterm birth, however, has limited efficacy and lacks preclinical data regarding mechanisms of action. It is unknown if nifedipine affects the pro-inflammatory environment associated with preterm labour pathophysiology and we hypothesise nifedipine only targets myometrial contraction rather than also mitigating inflammation. We assessed anti-inflammatory and anti-contractile effects of nifedipine on human myometrium using in vitro and ex vivo techniques, and a mouse model of preterm birth. We show that nifedipine treatment inhibited contractions in myometrial in vitro contraction assays (P = 0.004 vs. vehicle control) and potently blocked spontaneous and oxytocin-induced contractions in ex vivo myometrial tissue in muscle myography studies (P = 0.01 vs. baseline). Nifedipine treatment did not reduce gene expression or protein secretion of pro-inflammatory cytokines in either cultured myometrial cells or ex vivo tissues. Although nifedipine could delay preterm birth in some mice, this was not consistent in all dams and was overall not statistically significant. Our data suggests nifedipine does not modulate preterm birth via inflammatory pathways in the myometrium, and this may account for its limited clinical efficacy. [ABSTRACT FROM AUTHOR]

Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2) transcripts are elevated in the circulation of individuals whose pregnancies are complicated by preterm fetal growth restriction (FGR). In this paper, we show that the cases with preeclampsia (PE) have increased circulating NR4A2 transcripts compared to those with normotensive FGR. We aimed to establish whether the dysfunctional placenta mirrors the increase in NR4A2 transcripts and further, to uncover the function of placental NR4A2. NR4A2 expression was detected in preterm and term placental tissue; expressed higher at term. NR4A2 mRNA expression and protein were not altered in placentas from preterm FGR or PE pregnancies. Hypoxia (1% O2 compared to 8% O2) significantly reduced cytotrophoblast NR4A2 mRNA expression, but not placental explant NR4A2 expression. Silencing cytotrophoblast NR4A2 expression under hypoxia (via short interfering (si)RNAs) did not alter angiogenic Placental Growth Factor, nor anti-angiogenic sFlt-1 mRNA expression or protein secretion, but increased expression of cellular antioxidant, oxidative stress, inflammatory, and growth genes. NR4A2 expression was also not altered in a model of tumour necrosis factor-α-induced endothelial dysfunction, or with pravastatin treatment. Further studies are required to identify the origin of the circulating transcripts in pathological pregnancies, and investigate the function of placental NR4A2. [ABSTRACT FROM AUTHOR]

Introduction: Poor gestational outcomes due to placental insufficiency can have lifelong consequences for mother and child. Objective: There is a need for better methods of diagnosis, and elemental metabolomics may provide a means to determine the risk of gestational disorders. Methods: This study used blood plasma samples collected at 36 weeks' gestation from women who later developed preeclampsia (n = 38), or small-for-gestational age babies (n = 91), along with matched controls (n = 193). Multi-element analysis was conducted by inductively coupled plasma mass spectrometer (ICP-MS), allowing simultaneous measurement of 28 elements. Results: Women who later developed PE, exhibited significantly increased concentrations of K, Rb and Ba. For SGA pregnancies, there was a significant increase in Cu and a decrease in As concentrations. Despite significant differences in single elements, the elemental profile of groups indicated no clustering of control, PE, or SGA samples. Positive predicative values correctly identified approximately 60% of SGA and 70% of PE samples. Conclusion: This is the first-time elemental metabolomics has been used to predict SGA and PE at 36 weeks. Though significant changes were identified, routine clinical use may be limited but may contribute to a multi marker test. Future analysis should include other biomarkers, metabolic data or clinical measurements made throughout gestation. [ABSTRACT FROM AUTHOR]

Previously, we identified increased maternal circulating DAAM2 mRNA in pregnancies complicated by preterm fetal growth restriction (FGR). Here, we assessed whether circulating DAAM2 mRNA could detect FGR, and whether the DAAM2 gene, known to play roles in the Wnt signalling pathway is expressed in human placenta and associated with dysfunction and FGR. We performed linear regression analysis to calculate area under the ROC curve (AUC) for DAAM2 mRNA expression in the maternal circulation of pregnancies complicated by preterm FGR. DAAM2 mRNA expression was assessed across gestation by qPCR. DAAM2 protein and mRNA expression was assessed in preterm FGR placenta using western blot and qPCR. DAAM2 expression was assessed in term cytotrophoblasts and placental explant tissue cultured under hypoxic and normoxic conditions by qPCR. Small interfering RNAs were used to silence DAAM2 in term primary cytotrophoblasts. Expression of growth, apoptosis and oxidative stress genes were assessed by qPCR. Circulating DAAM2 mRNA was elevated in pregnancies complicated by preterm FGR [p < 0.0001, AUC = 0.83 (0.78–0.89)]. Placental DAAM2 mRNA was detectable across gestation, with highest expression at term. DAAM2 protein was increased in preterm FGR placentas but demonstrated no change in mRNA expression. DAAM2 mRNA expression was increased in cytotrophoblasts and placental explants under hypoxia. Silencing DAAM2 under hypoxia decreased expression of pro-survival gene, BCL2 and oxidative stress marker, NOX4, whilst increasing expression of antioxidant enzyme, HMOX-1. The increased DAAM2 associated with FGR and hypoxia implicates a potential role in placental dysfunction. Decreasing DAAM2 may have cytoprotective effects, but further research is required to elucidate its role in healthy and dysfunctional placentas. [ABSTRACT FROM AUTHOR]

Placental insufficiency can cause fetal growth restriction and stillbirth. There are no reliable screening tests for placental insufficiency, especially near-term gestation when the risk of stillbirth rises. Here we show a strong association between low circulating plasma serine peptidase inhibitor Kunitz type-1 (SPINT1) concentrations at 36 weeks' gestation and low birthweight, an indicator of placental insufficiency. We generate a 4-tier risk model based on SPINT1 concentrations, where the highest risk tier has approximately a 2-5 fold risk of birthing neonates with birthweights under the 3rd, 5th, 10th and 20th centiles, whereas the lowest risk tier has a 0-0.3 fold risk. Low SPINT1 is associated with antenatal ultrasound and neonatal anthropomorphic indicators of placental insufficiency. We validate the association between low circulating SPINT1 and placental insufficiency in two other cohorts. Low circulating SPINT1 is a marker of placental insufficiency and may identify pregnancies with an elevated risk of stillbirth. There are no reliable tests for placental insufficiency, which can lead to fetal growth restriction and stillbirth. Here the authors demonstrate that low levels of circulating SPINT1 are associated to low birthweight, and several ultrasound and neonatal anthropomorphic indicators of placental insufficiency. [ABSTRACT FROM AUTHOR]

Preeclampsia is a hypertensive disorder of pregnancy, responsible for over 60 000 maternal deaths annually. Endothelial dysfunction is a central aspect to its pathophysiology, and currently, no medical therapeutic is available for its treatment. In this study, we aim to investigate the effect of epidermal growth factor (EGF) on endothelial dysfunction using primary human tissues. We performed a number of in vitro assays that mimic the vascular endothelial dysfunction that occurs in preeclampsia. Epidermal growth factor reduced the expression of vascular cell adhesion molecule-1, a marker of endothelial dysfunction, after insult with tumor necrosis factor α (TNF-α) or serum from women with preeclampsia. Additionally, after TNF-α insult, EGF reduced tube disruption and the adhesion of monocytes to primary human umbilical vein endothelial cells (HUVECs). Our findings suggest that EGF reduces endothelial dysfunction in primary HUVECs. Epidermal growth factor may have potential as a novel peptide treatment for preeclampsia and other diseases where there is endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are major obstetric health problems. Higher levels of T-helper (Th) 1 (proinflammatory) cytokines have been observed in pregnancies complicated with PE and IUGR; this is in contrast to the predominant Th2 (anti-inflammatory) cytokine environment found in uncomplicated pregnancies. Myostatin is best known as a negative regulator of muscle development and reportedly has a role in fat deposition, glucose metabolism, and cytokine modulation (outside the placenta). Myostatin concentrations in plasma and protein expression in placental tissue are significantly higher in women with PE. Expression of myostatin in IUGR and PE-IUGR and the effect of this protein on the cytokine production from the placenta is unknown. In the current study, significant differences were identified in the expression of myostatin in pregnancies complicated with IUGR, PE, and PE with IUGR. Furthermore, cytokine production by first-trimester placental tissues was altered following myostatin treatment. [ABSTRACT FROM AUTHOR]

Background: Preterm prelabor rupture of the fetal membranes (PPROM) is a significant contributor to the morbidity and mortality of preterm birth, particularly in the setting of chorioamnionitis. No sensitive or specific diagnostic or predictive test currently exists for the accurate diagnosis of chorioamnionitis. Our aim was to measure messenger RNA (mRNA) coding cytokines in the maternal blood and examine whether they were increased in association with chorioamnionitis at delivery. Methods/Results: We performed a prospective cohort study of women recruited with PPROM at a mean gestational age of 28.9 weeks at risk of developing chorioamnionitis. Blood was sampled from participants, and the expression of mRNA coding for proinflammatory genes was measured in women with and without chorioamnionitis at the time of delivery as well as gestation-matched healthy controls. Expression was measured using quantitative polymerase chain reaction (PCR) and also digital PCR. Interleukin 1β (IL1B) mRNA expression in maternal blood was elevated in women with chorioamnionitis compared to gestation-matched controls. Importantly, among women admitted with PPROM, digital PCR confirmed a significant increase in IL1B expression in maternal blood in women with chorioamnionitis compared to women without chorioamnionitis. Polymerase chain reaction array revealed that CD14, nuclear factor of κ light polypeptide gene enhancer in B-cells 1 (NFKB1), and tumor necrosis factor receptor super family-interacting serine-threonine kinase 1 mRNA were significantly increased in women with chorioamnionitis compared to controls. Digital PCR confirmed that NFKB1 mRNA was significantly increased in patients with chorioamnionitis compared to controls and that CD14 levels increased over time in patients with PPROM having chorioamnionitis. Conclusion: Measuring circulating proinflammatory mRNA in women with PPROM may distinguish those with chorioamnionitis from those without, in turn providing better targeted therapies and appropriate timing of delivery. [ABSTRACT FROM AUTHOR]

Candidate stem/progenitor cells have been identified in mouse endometrium as label-retaining cells (LRCs). The role of endometrial stem/progenitor cells in initiating estrogen-stimulated endometrial growth in prepubertal and cycling mice was investigated following a single 17β-estradiol (E2) injection in bromodeoxyuridine (BrdU)-labeled and -chased (LRC), ovariectomised mice. Proliferating (BrdU+/Ki-67+) and mitotic (BrdU+/PH3+) epithelial LRCs were first detected in prepubertal mice 8 hours following E2 treatment, initiating the proliferative response. In contrast, all epithelial LRCs and 16% of epithelial cells in cycling mice proliferated within 2 hours. In cycling mice, 12% of stromal LRCs initiated a proliferative response 8 hours after E2. Proliferating epithelial LRCs and most stromal LRCs (85%) lacked estrogen receptor-α (ESR1). These findings suggest that endometrial epithelial LRCs function as stem/progenitor cells by receiving proliferative signals from neighboring ESR1+ niche cells to initiate the growth of the epithelium during development, while mature epithelial cells may undergo self-replication in cycling endometrium. [ABSTRACT FROM PUBLISHER]

The contribution of the high abundance of inflammatory cells present in the human endometrium prior to and during menstruation is unknown with respect to endometrial repair and/or menstruation. In this study, the presence and localisation of markers for key inflammatory cells have been examined in a mouse model of endometrial breakdown and repair and the functional contribution of neutrophils has been determined. In the model, decidualisation is artificially induced and progesterone support withdrawn; the endometrial tissue progressively breaks down by 24 h after progesterone withdrawal and, by 48 h, has usually undergone complete repair. Neutrophils have been identified in low abundance in decidual tissue, rise in number during breakdown and are most abundant during early repair. Macrophages are barely detectable during breakdown or repair in this model, whereas uterine natural killer cells are found only in intact decidua. The functional contribution of neutrophils to endometrial breakdown and repair has been assessed via neutrophil depletion by using the antibody RB6-8C5. This antibody significantly depletes neutrophils from the circulation and tissue, affects endometrial breakdown and markedly delays endometrial repair. This study has therefore demonstrated that neutrophils are the most abundant leucocyte in this model and that they play an important functional role in the processes of endometrial breakdown and repair. [ABSTRACT FROM AUTHOR]

Preeclampsia is a pregnancy complication associated with elevated placental secretion of anti-angiogenic factors, maternal endothelial dysfunction and organ injury. GATA2 is a transcription factor expressed in the endothelium which regulates vascular homeostasis by controlling transcription of genes and microRNAs, including endothelial miR126. We assessed GATA2 and miR126 in preeclampsia. Whole blood circulating GATA2 mRNA and miR126 expression were significantly decreased in women with established early-onset preeclampsia compared to gestation-matched controls (p = 0.002, p < 0.0001, respectively). Using case-control groups selected from a large prospective cohort, whole blood circulating GATA2 mRNA at both 28 and 36 weeks' gestation was significantly reduced prior to the clinical diagnosis of preeclampsia (p = 0.012, p = 0.015 respectively). There were no differences in GATA2 mRNA or protein expression in preeclamptic placentas compared to controls, suggesting the placenta is an unlikely source. Inducing endothelial dysfunction in vitro by administering either tumour necrosis factor-α or placenta-conditioned media to endothelial cells, significantly reduced GATA2 mRNA expression (p < 0.0001), suggesting the reduced levels of circulating GATA2 mRNA may be of endothelial origin. Circulating GATA2 mRNA is decreased in women with established preeclampsia and decreased up to 12 weeks preceding onset of disease. Circulating mRNAs of endothelial origin may be a novel source of biomarker discovery for preeclampsia. [ABSTRACT FROM AUTHOR]