Progestin-only long-acting reversible-contraceptive (pLARC)-exposed endometria displays decidualized human endometrial stromal cells (HESCs) and hyperdilated thin-walled fragile microvessels. The combination of fragile microvessels and enhanced tissue factor levels in decidualized HESCs generates excess thrombin, which contributes to abnormal uterine bleeding (AUB) by inducing inflammation, aberrant angiogenesis, and proteolysis. The- zinc finger and BTB domain containing 16 (ZBTB16) has been reported as an essential regulator of decidualization. Microarray studies have demonstrated that ZBTB16 levels are induced by medroxyprogesterone acetate (MPA) and etonogestrel (ETO) in cultured HESCs. We hypothesized that pLARC-induced ZBTB16 expression contributes to HESC decidualization, whereas prolonged enhancement of ZBTB16 levels triggers an inflammatory milieu by inducing pro-inflammatory gene expression and tissue-factor-mediated thrombin generation in decidualized HESCs. Thus, ZBTB16 immunostaining was performed in paired endometria from pre- and post-depo-MPA (DMPA)-administrated women and oophorectomized guinea pigs exposed to the vehicle, estradiol (E2), MPA, or E2 + MPA. The effect of progestins including MPA, ETO, and levonorgestrel (LNG) and estradiol + MPA + cyclic-AMP (E2 + MPA + cAMP) on ZBTB16 levels were measured in HESC cultures by qPCR and immunoblotting. The regulation of ZBTB16 levels by MPA was evaluated in glucocorticoid-receptor-silenced HESC cultures. ZBTB16 was overexpressed in cultured HESCs for 72 h followed by a ± 1 IU/mL thrombin treatment for 6 h. DMPA administration in women and MPA treatment in guinea pigs enhanced ZBTB16 immunostaining in endometrial stromal and glandular epithelial cells. The in vitro findings indicated that: (1) ZBTB16 levels were significantly elevated by all progestin treatments; (2) MPA exerted the greatest effect on ZBTB16 levels; (3) MPA-induced ZBTB16 expression was inhibited in glucocorticoid-receptor-silenced HESCs. Moreover, ZBTB16 overexpression in HESCs significantly enhanced prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1), and tissue factor (F3) levels. Thrombin-induced interleukin 8 (IL-8) and prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA levels in control-vector-transfected HESCs were further increased by ZBTB16 overexpression. In conclusion, these results supported that ZBTB16 is enhanced during decidualization, and long-term induction of ZBTB16 expression by pLARCs contributes to thrombin generation through enhancing tissue factor expression and inflammation by enhancing IL-8 and PTGS2 levels in decidualized HESCs.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1β-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

Zika virus (ZIKV) is spread by mosquito bites or via sexual or vertical transmission. ZIKV-infected adults are generally asymptomatic, but can display mild symptoms including fever, joint pain, rash and conjunctivitis. However, during pregnancy, vertical ZIKV transmission can cause placental dysfunction and elicit severe fetal defects, including microcephaly, retinopathy, fetal growth restriction and/or stillbirth. Since no FDA-approved vaccine or anti-viral agents are currently available, ZIKV infection poses a global maternal-fetal health challenge. The maternal-fetal interface consists of maternal decidual and immune cells as well as fetal-derived trophoblasts. Compared to other cell types at the maternal-fetal interface, syncytiotrophoblasts, which form the outer layer of floating villi, are less-permissive to ZIKV, thereby preventing ZIKV transmission to the underlying cytotrophoblasts and/or other cells such as Hofbauer cells or fetal endothelium in the villi. However, anchoring villi are tightly attached to the decidua and their cytotrophoblastic cell columns are ZIKV-permissive, suggesting this location as the most likely site of ZIKV vertical transmission. Thus, at the maternal-fetal interface, maternal decidual cells likely serve as a reservoir of ZIKV persistence since they: 1) overexpress viral entry molecules compared to trophoblasts; 2) are highly permissive to ZIKV infection in a gestational age-dependent manner (more easily infected earlier in gestation); 3) augment ZIKV infection of weakly permissive primary cytotrophoblast cultures; and 4) display local maternal-immune tolerance, which prolongs ZIKV survival to facilitate fetal transmission. This review focuses on molecular mechanisms underlying ZIKV infection of cells at the human maternal-fetal interface, thus highlighting how decidual cells enhance propagation of ZIKV in extravillous cytotrophoblasts and why development of agents that eliminate ZIKV persistence in reproductive tissues before pregnancy is crucial to prevent perinatal ZIKV transmission.

SARS-CoV-2 infects cells via binding to ACE2 and TMPRSS2, which allows the virus to fuse with host cells. The viral RNA is detected in the placenta of SARS-CoV-2-infected pregnant women and infection is associated with adverse pregnancy complications. Therefore, we hypothesize that SARS-CoV-2 infection of placental cells induces pro-inflammatory cytokine release to contribute to placental dysfunction and impaired pregnancy outcomes. First, expression of ACE2 and TMPRSS2 was measured by qPCR in human primary cultured term cytotrophoblasts (CTBs), syncytiotrophoblast (STBs), term and first trimester decidual cells (TDCs and FTDCs, respectively), endometrial stromal cells (HESCs) as well as trophoblast cell lines HTR8, JEG3, placental microvascular endothelial cells (PMVECs) and endometrial endothelial cells (HEECs). Later, cultured HTR8, JEG3, PMVECs and HEECs were treated with 10, 100, 1000 ng/ml of recombinant (rh-) SARS-CoV-2 S-protein ± 10 ng/ml rh-IFNγ. Pro-inflammatory cytokines IL-1β, 6 and 8, chemokines CCL2, CCL5, CXCL9 and CXCL10 as well as tissue factor (F3), the primary initiator of the extrinsic coagulation cascade, were measured by qPCR as well as secreted IL-6 and IL-8 levels were measured by ELISA. Immunohistochemical staining for SARS-CoV-2 spike protein was performed in placental specimens from SARS-CoV-2–positive and normal pregnancies. ACE2 levels were significantly higher in CTBs and STBs vs. TDCs, FTDCs and HESCs, while TMPRSS2 levels were not detected in TDCs, FTDCs and HESCs. HTR8 and JEG3 express ACE2 and TMPRSS2, while PMVECs and HEECs express only ACE2, but not TMPRSS2. rh-S-protein increased proinflammatory cytokines and chemokines levels in both trophoblast and endothelial cells, whereas rh-S-protein only elevated F3 levels in endothelial cells. rh-IFNγ ± rh-S-protein augments expression of cytokines and chemokines in trophoblast and endothelial cells. Elevated F3 expression by rh-IFNγ ± S-protein was observed only in PMVECs. In placental specimens from SARS-CoV-2-infected mothers, endothelial cells displayed higher immunoreactivity against spike protein. These findings indicated that SARS-CoV-2 infection in placental cells: 1) induces pro-inflammatory cytokine and chemokine release, which may contribute to the cytokine storm observed in severely infected pregnant women and related placental dysfunction; and 2) elevates F3 expression that may trigger systemic or placental thrombosis.

To determine if uterine artery flow volume (UtaV) estimated throughout gestation is different in pregnancies complicated by SGA, pre-eclampsia (PE), or adverse neonatal outcomes; to evaluate the potential of using UtaV as a screening tool when combined with maternal characteristics. This prospective longitudinal study over a 5-year period measured uterine artery vessel diameter and time-averaged maximum velocity (TAMXV) using color Doppler between 11+0 to 14+0, 18+0 to 22+6, and 28+0 to 34+6 weeks of gestation. UtaV was calculated from the vessel diameter and TAMXV. SGA was defined as birthweight < 10th percentile. PE was defined using ACOG criteria. A composite adverse neonatal outcome (ANO) was defined including presence of any of the following: 5 min Apgar score <5, arterial pH <7.10, respiratory distress syndrome, grade III or IVH (III or IV), seizures or neonatal death. Student t-tests were used to compared UtaV between normal and complicated pregnancies. ROC curves were constructed and AUCs compared. 397 pregnancies were included. Among pregnancies evaluated, SGA was seen in 26 (6.5%), PE in 22 (5.7%) and ANO in 22 (3.5%). The UtaV increased with gestational age (Figure). UtaV was significantly lower in SGA neonates in all trimesters but not in ANO (Table). UtaV was only significantly different in PE in the third-trimester. This difference persisted when UtaV was corrected for estimated fetal weight at the time of estimation (p=0.02). Prediction models for SGA including maternal characteristics: race, body mass index, smoking, hypertension, pre-gestational diabetes and UtaV had areas under the ROC curves of 0.68, 0.69 and 0.71 in first, second, and third trimesters, respectively. For PE, the AUC was 0.83, 0.82, 0.81, respectively. Uterine artery blood flow in all trimesters is reduced in pregnancies resulting in SGA. In pregnancies resulting in PE, UtaV was reduced in the third trimester. When combined with maternal characteristics, UtaV represents a promising screening marker for SGA and PE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Context Enhanced levels of catecholestradiols, 2-hydroxyestradiol (2-OHE2) or 4-hydroxyestradiol (4-OHE2), are reported in endometriosis. During gestation, catecholestradiol activation of adrenergic receptors (AR) elevates estrogen receptor (ER)-independent proliferation of uterine arterial endothelial cells. Objective To investigate beta-AR-mediated catecholestradiol effects on human endometrial stromal cell (HESC) and epithelial cell survival in endometriosis. Design beta-AR immunostaining of eutopic and ectopic endometria (n = 9). Assays for cell viability, 5-bromo-2 '-deoxyuridine proliferation, apoptosis, quantitative PCR, and estrogenicity (alkaline phosphatase activity), as well as siRNA beta-AR silencing and immunoblot analyses of cultured HESCs or Ishikawa cells treated with control or 2-OHE2 or 4-OHE2 +/-beta-AR antagonist or +/- p38 MAPK inhibitor. Setting University research institution. Patients Women with or without endometriosis. Interventions None. Main Outcome Measures beta-AR expression in eutopic vs ectopic endometria and regulation of HESC survival by 2-OHE2 and 4-OHE2. Results Eutopic and ectopic endometrial stromal and epithelial cells displayed beta 2-AR immunoreactivity with increased staining in the functionalis vs basalis layer (P < 0.05). Both 2-OHE2 and 4-OHE2 enhanced HESC and Ishikawa cell survival (P < 0.05), an effect abrogated by beta-AR antagonist propranolol, but not ER antagonist ICI182,780. 2-OHE2 or 4-OHE2 failed to induce cell survival and estrogenic activity in ADRB2-silenced HESCs and in Ishikawa cells, respectively. Although 2-OHE2 inhibited apoptosis and BAX mRNA expression, 4-OHE2 induced proliferation and decreased apoptosis (P < 0.05). Both catecholestradiols elevated phospho-p38 MAPK levels (P < 0.05), which was blocked by propranolol, and p38 MAPK inhibitor reversed catecholestradiol-enhanced HESC survival. Conclusions Catecholestradiols increase endometrial cell survival by an ER-independent beta-AR-mediated p38 MAPK activation, suggesting that agents blocking beta-AR (e.g., propranolol) or inhibiting 2-OHE2- or 4-OHE2-generating enzymes (i.e., CYP1A1/B1) could treat endometriosis.

To determine if umbilical vein flow (QuV) estimated throughout gestation is different in pregnancies resulting in SGA or adverse neonatal outcomes and evaluate the potential of using this as a screening tool when combined with maternal characteristics. This prospective longitudinal study over a 5-year period measured umbilical vein diameter and time-averaged maximum velocity (TAMXV)using Color Doppler between 11 to 13 + 6, 18 to 21 + 6 and 28 to 34 weeks of gestation. QuV was calculated from the vein diameter and TAMXV. SGA was defined as birthweight < 10th percentile. QuV was not adjusted for estimated fetal weight as the later is in the causal pathway leading to SGA. A composite adverse neonatal outcome (ANO) was defined including presence of any of the following: 5 min Apgar score <5, arterial pH <7.10, respiratory distress syndrome, grade III or IVH (III or IV), seizures or neonatal death. Student t-tests were used to compared QuV between normal and complicated pregnancies. ROC curves were constructed and AUCs compared. Among 537 pregnancies evaluated, SGA was seen in 38 (7.1%) and ANO in 22 (4.1%).The QuV increased with gestational age (Figure). QuV and QuV adjusted for EFW were significantly lower in SGA and ANO groups in the second trimester. In the third-trimester, absolute QuV was significantly different only in pregnancies complicated by SGA. Prediction models for SGA including maternal characteristics: race, body mass index, smoking, hypertension, pre-gestational diabetes and QuV had areas under the ROC curves of, 0.69, 0.71 and 0.72 in first, second and third, trimesters respectively. Umbilical vein blood flow is reduced in pregnancies resulting in SGA and adverse neonatal outcomes and represents a promising screening marker when combined with maternal characteristics.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Superb Micovascular Imaging (SMI) improves visualization of small vessels by suppressing global motions while preserving low-flow components such as the micro-vessels in the placenta. We sought to determine if SMI aided visualization of flow velocities in spiral artery (SA) and the intravillous space (IV) can predict development of late onset fetal growth restriction (FGR), gestational hypertension (GHT) and/or preeclampsia (PE). Prospective longitudinal study measuring SA and IV flow velocity using SMI Color Doppler between 11 to 13 + 6, 18 to 21 + 6 and 28 to 34 weeks of gestation. SA and IV flow velocities were reported as resistance indices (RI). Late FGR was defined as EFW < 10th percentile after 34 weeks, and GHT and PE by ACOG standards. RI values were analyzed with multilevel modeling: individual regression curves were estimated and combined to obtain the reference intervals for SA and IV flow velocities in normal pregnancies. Student t-tests compared deviation from expected flow velocity between normal and complicated pregnancies. Of 540 pregnancies included; 19 (3.5%) had FGR, 31 (5.7%) PE and 61 (11.3%) GHT. In uncomplicated pregnancies, SA RI decreased progressively with advancing gestational age, while IV RI decreased with gestational age (Fig). Mean RI values for both SA and IV were significantly higher in the FGR group compared with uncomplicated pregnancies in the third trimester (Table). There was no significant difference in mean RI between uncomplicated pregnancies and those with PE and GHT at any gestational age. When all adverse outcomes were combined, increased SA RI was significantly associated with these outcomes only in the third trimester when compared with the control group [mean RI (SD)= 0.27(0.11) versus 0.33(0.11), respectively; p=0.042]. SMI improved the visualization of SA and IV. Significant differences were seen in pregnancies at risk mostly for late-onset FGR, but only in the early third-trimester.The potential role of SA and IV for predicting adverse outcomes appear limited. Supported by U01HD087213.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Depression and posttraumatic stress disorder increase the risk of idiopathic preterm birth (iPTB); however, the exact molecular mechanism is unknown. Depression and stress-related disorders are linked to increased FK506-binding protein 51 (FKBP51) expression levels in the brain and/or FKBP5 gene polymorphisms. Fkbp5-deficient (Fkbp5(-/-)) mice resist stress-induced depressive and anxiety-like behaviors. FKBP51 binding to progesterone (P4) receptors (PRs) inhibits PR function. Moreover, reduced PR activity and/or expression stimulates human labor. We report enhanced in situ FKBP51 expression and increased nuclear FKBP51-PR binding in decidual cells of women with iPTB versus gestational age-matched controls. In Fkbp5(+/+) mice, maternal restraint stress did not accelerate systemic P4 withdrawal but increased Fkbp5, decreased PR, and elevated AKR1C18 expression in uteri at E17.25 followed by reduced P4 levels and increased oxytocin receptor (Oxtr) expression at 18.25 in uteri resulting in PTB. These changes correlate with inhibition of uterine PR function by maternal stress-induced FKBP51. In contrast, Fkbp5(-/-) mice exhibit prolonged gestation and are completely resistant to maternal stress-induced PTB and labor-inducing uterine changes detected in stressed Fkbp5(+/+) mice. Collectively, these results uncover a functional P4 withdrawal mechanism mediated by maternal stress-induced enhanced uterine FKBP51 expression and FKPB51-PR binding, resulting in iPTB.