Extracellular vesicles secreted by human uterine stromal cells regulate decidualization, angiogenesis, and trophoblast differentiation.

In humans, the uterus undergoes a dramatic transformation to form an endometrial stroma-derived secretory tissue, termed decidua, during early pregnancy. The decidua secretes various factors that act in an autocrine/paracrine manner to promote stromal differentiation, facilitate maternal angiogenesis, and influence trophoblast differentiation and development, which are critical for the formation of a functional placenta. Here, we investigated the mechanisms by which decidual cells communicate with each other and with other cell types within the uterine milieu. We discovered that primary human endometrial stromal cells (HESCs) secrete extracellular vesicles (EVs) during decidualization and that this process is controlled by a conserved HIF2α-RAB27B pathway. Mass spectrometry revealed that the decidual EVs harbor a variety of protein cargo, including cell signaling molecules, growth modulators, metabolic regulators, and factors controlling endothelial cell expansion and remodeling. We tested the hypothesis that EVs secreted by the decidual cells mediate functional communications between various cell types within the uterus. We demonstrated that the internalization of EVs, specifically those carrying the glucose transporter 1 (GLUT1), promotes glucose uptake in recipient HESCs, supporting and advancing the decidualization program. Additionally, delivery of HESC-derived EVs into human endothelial cells stimulated their proliferation and led to enhanced vascular network formation. Strikingly, stromal EVs also promoted the differentiation of trophoblast stem cells into the extravillous trophoblast lineage. Collectively, these findings provide a deeper understanding of the pleiotropic roles played by EVs secreted by the decidual cells to ensure coordination of endometrial differentiation and angiogenesis with trophoblast function during the progressive phases of decidualization and placentation.

Neurotrophins and Their Receptors, Novel Therapeutic Targets for Pelvic Pain in Endometriosis, Are Coordinately Regulated by IL-1ß via the JNK Signaling Pathway.

Pelvic pain in women with endometriosis is attributed to neuroinflammation and afferent nociceptor nerves in ectopic and eutopic endometrium. The hypothesis that uterine nociception is activated by IL-1ß, a prominent cytokine in endometriosis, was tested herein. Immunofluorescence histochemistry confirmed the presence of neurons in human endometrial tissue. Expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and their receptors in endometrial tissue and cells was validated by immunohistochemistry and Western blotting. Isolated endometrial stromal cells (ESCs) were subjected to dose-response and time-course experiments with IL-1ß and kinase inhibitors to characterize in vitro biomarkers. Neural biomarkers were co-localized in endometrial nerve fibers. NGF, BDNF, and their receptors tropomyosin receptor kinase (Trk) A, TrkB, and p75 neurotrophin receptor were all expressed in primary ESCs. IL-1ß stimulated higher TrkA/B expression in ESCs derived from endometriosis cases (2.8- ± 0.2-fold) than cells from controls (1.5- ± 0.3-fold, t-test, P < 0.01), effects that were mediated via the c-Jun N-terminal kinase (JNK) pathway. BDNF concentrations trended higher in peritoneal fluid of endometriosis cases but were not statistically different from controls (P = 0.16). The results support the hypothesis that NGF and BDNF and their corresponding receptors orchestrate innervation of the endometrium, which is augmented by IL-1ß. We postulate that JNK inhibitors, such as SP600125, have the potential to reduce neuroinflammation in women with endometriosis.

Association between maternal cigarette smoking cessation and risk of preterm birth in Western New York.

BACKGROUND: Although many studies suggested the benefit of smoking cessation among pregnant women in reducing the risk of preterm birth (PTB), the timing of the effect of the cessation remains inconclusive. OBJECTIVES: To examine the association of trimester-specific smoking cessation behaviours with PTB risk. METHODS: We included 199,453 live births in Western New York between 2004 and 2018. Based on self-reported cigarette smoking during preconception and in each trimester, we created six mutually exclusive groups: non-smokers, quitters in each trimester, those who smoked throughout pregnancy, and inconsistent smokers. Risk ratios (RRs) and 95% confidence intervals (CIs) were estimated using Poisson regression to examine the association between smoking cessation and PTB. Effect modification by illegal drug use, maternal age, race and ethnicity and pre-pregnancy body mass index (BMI) was investigated multiplicatively by ratio of relative risk and additively by relative excess risk due to interaction (RERI). RESULTS: Overall, 6.7% of women had a PTB; 14.1% smoked throughout pregnancy and 3.4%, 1.8% and 0.8% reported quitting smoking during the first, second and third trimesters, respectively. Compared to non-smokers, third-trimester cessation (RR 1.20, 95% CI 1.01, 1.43) and smoking throughout pregnancy (RR 1.27, 95% CI 1.21, 1.33) were associated with a higher PTB risk, while quitting smoking during the first or second trimester, or inconsistent smoking was not associated with PTB. A positive additive interaction was identified for maternal age and late smoking cessation or smoking throughout pregnancy on PTB risk (RERI 0.17, 95% CI 0.00, 0.36), and a negative interaction was observed for pre-pregnancy BMI ≥30 kg/m2 (ratio of relative risk 0.70, 95% CI 0.63, 0.78; RERI -0.42, 95% CI -0.56, -0.30). CONCLUSION: Compared to non-smokers, smoking throughout pregnancy and third-trimester smoking cessation are associated with an increased risk of PTB, while quitting before the third trimester may not increase PTB risk.

A hypoxia-induced Rab pathway regulates embryo implantation by controlled trafficking of secretory granules.

Implantation is initiated when an embryo attaches to the uterine luminal epithelium and subsequently penetrates into the underlying stroma to firmly embed in the endometrium. These events are followed by the formation of an extensive vascular network in the stroma that supports embryonic growth and ensures successful implantation. Interestingly, in many mammalian species, these processes of early pregnancy occur in a hypoxic environment. However, the mechanisms underlying maternal adaptation to hypoxia during early pregnancy remain unclear. In this study, using a knockout mouse model, we show that the transcription factor hypoxia-inducible factor 2 alpha (Hif2α), which is induced in subluminal stromal cells at the time of implantation, plays a crucial role during early pregnancy. Indeed, when preimplantation endometrial stromal cells are exposed to hypoxic conditions in vitro, we observed a striking enhancement in HIF2α expression. Further studies revealed that HIF2α regulates the expression of several metabolic and protein trafficking factors, including RAB27B, at the onset of implantation. RAB27B is a member of the Rab family of GTPases that allows controlled release of secretory granules. These granules are involved in trafficking MMP-9 from the stroma to the epithelium to promote luminal epithelial remodeling during embryo invasion. As pregnancy progresses, the HIF2α-RAB27B pathway additionally mediates crosstalk between stromal and endothelial cells via VEGF granules, developing the vascular network critical for establishing pregnancy. Collectively, our study provides insights into the intercellular communication mechanisms that operate during adaptation to hypoxia, which is essential for embryo implantation and establishment of pregnancy.

Adiposity and Endometriosis Severity and Typology.

STUDY OBJECTIVE: Prior research has collectively shown that endometriosis is inversely related to women's adiposity. The aim of this study was to assess whether this inverse relationship holds true by disease severity and typology. DESIGN: Cross-sectional study among women with no prior diagnosis of endometriosis. SETTING: Fourteen clinical centers in Salt Lake City, UT, and San Francisco, CA. PATIENTS: A total of 495 women (of which 473 were analyzed), aged 18-44 years, were enrolled in the operative cohort of the Endometriosis, Natural History, Diagnosis, and Outcomes (ENDO) Study. INTERVENTIONS: Gynecologic laparoscopy/laparotomy regardless of clinical indication. MEASUREMENTS AND MAIN RESULTS: Participants underwent anthropometric assessments, body composition measurements, and evaluations of body fat distribution ratios before surgery. Surgeons completed a standardized operative report immediately after surgery to capture revised American Society for Reproductive Medicine staging (I-IV) and typology of disease (superficial endometriosis [SE], ovarian endometrioma [OE], and deep infiltrating endometriosis [DIE]). Linear mixed models, taking into account within-clinical-center correlation, were used to generate least square means (95% confidence intervals) to assess differences in adiposity measures by endometriosis stage (no endometriosis, I-IV) and typology (no endometriosis, SE, DIE, OE, OE + DIE) adjusting for age, race/ethnicity, and parity. Although most confidence intervals were wide and overlapping, 3 general impressions emerged: (1) women with incident endometriosis had the lowest anthropometric/body composition indicators compared with those without incident endometriosis, (2) women with stage I or IV endometriosis had lower indicators compared with women with stage II or III, and (3) women with OE and/or DIE tended to have the lowest indicators, whereas women with SE had the highest indicators. CONCLUSION: Our research highlights that the relationship between women's adiposity and endometriosis severity and typology may be more complicated than prior research indicates.

Aberrant retinoic acid production in the decidua: Implications for pre-eclampsia.

Fine-tuning of the endometrium during the evanescent 'window of implantation' relies upon an array of diverse and redundant signaling molecules, particularly the ovarian steroids E2 and P4, but also growth factors, eicosanoids, and vitamins including the vitamin A compounds (retinoids). Pregnancy complications such as preeclampsia (PE) can result from aberrations in the production or function of these molecules that arise during this critical period of decidual development. Such aberrations may be reflected by incomplete decidualization, reduced spiral artery modification, and/or loss of immune tolerance to the developing fetus. Our understanding of the role of the active retinoid metabolite all-trans retinoic acid (RA) in maintaining immune balance in certain tissues, along with data describing its role in decidualization, present a compelling argument that aberrant RA signaling in the decidua can play a significant role in the etiology of PE. Recent findings that decidualization and expression of the anti-angiogenic gene product, 'soluble fms-like tyrosine kinase-1' (sFLT1) are negatively correlated and that sFLT1 expression is directly inhibited by RA, provide additional evidence of the critical role of this retinoid in regulating early vascular development in the decidua. This review provides insight into the production and function of RA in the decidua and how modifications in its metabolism and signaling might lead to certain pregnancy disorders such as PE.

Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF-κB signaling pathway.

Endometriosis is a chronic gynecological inflammatory disorder in which immune system dysregulation is thought to play a role in its initiation and progression. Due to altered sex steroid receptor concentrations and other signaling defects, eutopic endometriotic tissues have an attenuated response to progesterone. This progesterone-resistance contributes to lesion survival, proliferation, pain, and infertility. The current agency-approved hormonal therapies, including synthetic progestins, GnRH agonists, and danazol are often of limited efficacy and counterproductive to fertility and cause systemic side effects due to suppression of endogenous steroid hormone levels. In the current study, we examined the effects of curcumin (CUR, diferuloylmethane), which has long been used as an anti-inflammatory folk medicine in Asian countries for this condition. The basal levels of proinflammatory and proangiogenic chemokines and cytokines expression were higher in primary cultures of stromal cells derived from eutopic endometrium of endometriosis (EESC) subjects compared with normal endometrial stromal cells (NESC). The treatment of EESC and NESC with CUR significantly and dose-dependently reduced chemokine and cytokine secretion over the time course. Notably, CUR treatment significantly decreased phosphorylation of the IKKα/ß, NF-κB, STAT3, and JNK signaling pathways under these experimental conditions. Taken together, our findings suggest that CUR has therapeutic potential to abrogate aberrant activation of chemokines and cytokines, and IKKα/ß, NF-κB, STAT3, and JNK signaling pathways to reduce inflammation associated with endometriosis.

Interleukin-1ß inhibits estrogen receptor-α, progesterone receptors A and B and biomarkers of human endometrial stromal cell differentiation: implications for endometriosis.

Human blastocyst nidation in the uterus and successful pregnancy require coordinated endometrial expression of estrogen receptor (ER)-α, progesterone receptors (PR)-A and -B and the gap junction protein, connexin (Cx)43. Our prior work established that inflammation associated with conditions of reduced fecundity, particularly endometriosis, can perturb eutopic decidual function. In the current studies, we have modeled endometrial decidualization in primary human endometrial stromal cell cultures derived from normal controls (NESC) and from the eutopic endometria of women with endometriosis (EESC) to test the hypothesis that a proinflammatory cytokine, interleukin (IL)-1ß, can disrupt stromal cell differentiation. The cells were grown under a standard protocol with hormones (10 nM 17ß-estradiol, 100 nM progesterone and 0.5 mM dibutyryl cAMP) for up to 7 days in the absence or presence of IL-1ß. Time-course experiments showed that IL-1ß compromised decidual function in both NESC and EESC, which was accompanied by rapid phosphorylation of ER-α, PR and Cx43 and their cellular depletion. Inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway by a selective pharmacological blocker (PD98059) or siRNA interference, or the addition of hormones themselves, blocked the phosphorylation of ERK mediators; increased the production of steroid receptors, Cx43, prolactin, insulin-like growth factor binding protein-1 (IGFBP)-1 and vascular endothelial growth factor (VEGF) and accelerated the differentiation. The results indicate that inhibition of IL-1ß can enhance decidualization in NESC and EESC in vitro. Strategies to interfere with this pathway might be implemented as an in vivo approach to enhance fertility in women with endometriosis and, potentially, other inflammatory pathologies.

IL-1ß Stimulates Brain-Derived Neurotrophic Factor Production in Eutopic Endometriosis Stromal Cell Cultures: A Model for Cytokine Regulation of Neuroangiogenesis.

Endometriosis implants are comprised of glandular and stromal elements, macrophages, nerves, and blood vessels and are commonly accompanied by pelvic pain. We propose that activated macrophages are recruited to and infiltrate nascent lesions, where they secrete proinflammatory cytokines, promoting the production of chemokines, neurotrophins, and angiogenic growth factors that sustain an inflammatory microenvironment. Immunohistochemical evaluation of endometriosis lesions reveals in situ colocalization of concentrated macrophages, brain-derived neurotrophic factor (BDNF), and nerve fibers. These observations were coupled with biochemical analyses of primary eutopic endometriosis stromal cell (EESC) cultures, which allowed defining potential pathways leading to the neuroangiogenic phenotype of these lesions. Our findings indicate that IL-1ß potently (EC50 = 7 ± 2 ng/mL) stimulates production of EESC BDNF at the mRNA and protein levels in an IL-1 receptor-dependent fashion. Selective kinase inhibitors demonstrate that this IL-1ß effect is mediated by c-Jun N-terminal kinase (JNK), NF-κB, and mechanistic target of rapamycin signal transduction pathways. IL-1ß regulation of regulated on activation normal T cell expressed and secreted (RANTES), a prominent EESC chemokine, also relies on JNK and NF-κB. An important clinical implication of the study is that interference with BDNF and RANTES production, by selectively targeting the JNK and NF-κB cascades, may offer a tractable therapeutic strategy to mitigate the pain and inflammation associated with endometriosis.

Assessing research gaps and unmet needs in endometriosis.

Endometriosis, a systemic disease that is often painful and chronic, affects ∼10% of reproductive-age women. The disease can have a negative impact on a patient's physical and emotional well-being, quality of life, and productivity. Endometriosis also places significant economic and social burden on patients, their families, and society as a whole. Despite its high prevalence and cost, endometriosis remains underfunded and underresearched, greatly limiting our understanding of the disease and slowing much-needed innovation in diagnostic and treatment options. Due in part to the societal normalization of women's pain and stigma around menstrual issues, there is also a lack of disease awareness among patients, health care providers, and the public. The Society for Women's Health Research convened an interdisciplinary group of expert researchers, clinicians, and patients for a roundtable meeting to review the current state of the science on endometriosis and identify areas of need to improve a woman's diagnosis, treatment, and access to quality care. Comprehensive and interdisciplinary approaches to disease management and increased education and disease awareness for patients, health care providers, and the public are needed to remove stigma, increase timely and accurate diagnosis and treatment, and allow for new advancements.

Discovery and Characterization of Human Amniochorionic Membrane Microfractures.

This study obtained visual evidence of novel cellular and extracellular matrix-level structural alterations in term and preterm human fetal amniochorionic membranes. Amniochorions were collected from term cesarean (not in labor) or vaginal (labor) deliveries, preterm premature rupture of membranes, and spontaneous preterm birth. To determine the effect of oxidative stress on membranes at term or preterm labor, term not in labor samples in an organ explant culture in vitro were exposed to cigarette smoke extract. Tissues were imaged using multiphoton autofluorescence and second harmonic generation microscopy. Images were analyzed using ImageJ and IMARIS software. Three-dimensional microscopic analysis of membranes revealed microfractures that were characterized by amnion cell puckering, basement membrane degradation, and tunnels that extended into the collagen matrix with migrating cells. Numbers of microfractures were similar at term regardless of labor status; however, morphometric measures (width and depth) were higher in term labor membranes. Oxidative stress induced higher numbers of microfractures in term not in labor membranes, with morphometry resembling that seen in term labor membranes. Preterm premature rupture of the membranes had the highest number of microfractures compared to membranes from term and other preterm births. Microfractures are structural alterations indicative of areas of tissue remodeling during gestation. Their increase at preterm and in response to oxidative stress may indicate failure to reseal, predisposing membranes to rupture.

Amnion epithelial cell-derived exosomes induce inflammatory changes in uterine cells.

BACKGROUND: Fetal endocrine signals are generally considered to contribute to the timing of birth and the initiation of labor. Fetal tissues under oxidative stress release inflammatory mediators that lead to sterile inflammation within the maternal-fetal interface. Importantly, these inflammatory mediators are packaged into exosomes, bioactive cell-derived extra cellular vesicles that function as vectors and transport them from the fetal side to the uterine tissues where they deposit their cargo into target cells enhancing uterine inflammatory load. This exosome-mediated signaling is a novel mechanism for fetal-maternal communication. OBJECTIVE: This report tested the hypothesis that oxidative stress can induce fetal amnion cells to produce exosomes, which function as a paracrine intermediary between the fetus and mother and biochemically signal readiness for parturition. STUDY DESIGN: Primary amnion epithelial cells were grown in normal cell culture (control) or exposed to oxidative stress conditions (induced by cigarette smoke extract). Exosomes were isolated from cell supernatant by sequential ultracentrifugation. Exosomes were quantified and characterized based on size, shape, and biochemical markers. Myometrial, decidual, and placental cells (BeWo) were treated with 2 × 105, 2 × 107, and 2 × 109 control or oxidative stress-derived amnion epithelial cell exosomes for 24 hours. Entry of amnion epithelial cell exosomes into cells was confirmed by confocal microscopy of fluorescent-labeled exosomes. The effect of amnion epithelial cell exosomes on target cell inflammatory status was determined by measuring production of interleukin-6, interleukin-8, interleukin-1ß, tumor necrosis factor-α, and prostaglandin E2 by enzyme-linked immunosorbent assay and inflammatory gene transcription factor (nuclear factor-κß) activation status by immunoblotting for phosphorylated RelA/p65. Localization of NANOG in term human myometrium and decidua obtained from women before labor and during labor was performed using immunohistochemistry. Data were analyzed by Wilcoxon-Mann-Whitney test to compare effects of exosomes from control and oxidative stress-treated amnion epithelial cells on inflammatory status of target cells. RESULTS: Amnion epithelial cells released ∼125 nm, cup-shaped exosomes with ∼899 and 1211 exosomes released per cell from control and oxidative stress-induced cells, respectively. Amnion epithelial cell exosomes were detected in each target cell type after treatment using confocal microscopy. Treatment with amnion epithelial cell exosomes increased secretion of interleukin-6, interleukin-8, and PGE2 and activation of NF-κß (each P < .05) in myometrial and decidual cells. Exosome treatments had no effect on interleukin-6 and PGE2 production in BeWo cells. NANOG staining was higher in term labor myometrium and decidua compared to tissues not in labor. CONCLUSION: In vitro, amnion epithelial cell exosomes lead to an increased inflammatory response in maternal uterine cells whereas placental cells showed refractoriness. Fetal cell exosomes may function to signal parturition by increasing maternal gestational cell inflammation.

Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.

Progesterone resistance in endometriosis: origins, consequences and interventions.

Endometriosis is a common cause of pelvic pain and affects up to 10% of women of reproductive age. Aberrant progesterone signaling in the endometrium plays a significant role in impaired decidualization and establishment of ectopic endometrial implants. Eutopic endometrial cells from women with endometriosis fail to downregulate genes needed for decidualization, such as those involved in cell cycle regulation, leading to unbridled proliferation. Several causes of progesterone resistance in the endometrium have been postulated, including congenital "preconditioning", whereby the in utero environment renders infants susceptible to neonatal uterine bleeding and endometriosis. Progesterone action is crucial to decreasing inflammation in the endometrium, and deviant progesterone signaling results in a proinflammatory phenotype. Conversely, chronic inflammation can induce a progesterone-resistant state. Repetitive retrograde endometrial shedding begets chronic peritoneal inflammation, which further exacerbates progesterone resistance. Genetic causes of progesterone resistance include progesterone receptor gene polymorphisms, altered microRNA expression, and epigenetic modifications to progesterone receptors and their targets. Environmental toxins such as dioxin play a possible role in the genesis of endometriosis by permitting an inflammatory milieu. A consequence of impaired progesterone action is that hormonal therapy is rendered ineffective for a subset of women with endometriosis. Synthetic progestins, such as dienogest, may overcome this phenomenon by increasing progesterone receptor expression and decreasing proinflammatory cytokines. Other modalities include high dose depot formulations of progestins, medicated intrauterine devices and the likely advent of oral GnRH antagonists. Unearthing root causes of progesterone inaction in endometriosis will aid in the development of novel therapeutics geared toward prevention and treatment.

Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro.

Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson's theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.

Downregulation of apelin in the human placental chorionic villi from preeclamptic pregnancies.

The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36-38 wk of gestation. Total apelin content was lower in PRE compared with NORM chorionic villi (49.7±3.4 vs. 72.3±9.8 fmol/mg protein; n=20-22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2) activity was higher in PRE villi (572.0±23.0 vs. 485.3±24.8 pmol·mg(-1)·min(-1); n=18-22). A low dose of ANG II (1 nM; 2 h) decreased apelin release in NORM villous explants that was blocked by the ANG II receptor 1 (AT1) antagonist losartan. Moreover, losartan enhanced apelin release above the 2-h baseline levels in both NORM and PRE villi (P<0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by ANG II.

A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells.

Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (C x 43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on C x 43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of C x 43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of C x 43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of C x 43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.

Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro.

Preterm prelabor rupture of the membranes (pPROM) may lead to preterm births (PTBs). We investigated premature senescence of fetal membranes in women with pPROM and spontaneous PTB with intact membranes (<34 weeks) and the inducibility fetal membrane senescence phenotype by oxidative stress in vitro. IHC was performed for p53, p21, and phospho (p)-p38 mitogen-activated protein kinase (MAPK) as markers of senescence phenotype in pPROM, PTBs, and term births. Term fetal membranes were exposed to cigarette smoke extract to induce oxidative stress. Western blots documented p-p53 and p-p38 MAPK. Transmission electron microscopy assessed cellular morphologic features in clinical and cigarette smoke extract-treated membranes. A total of 80% of pPROM cells and >60% of term cells were positive for all three senescence phenotype markers, and concentrations were higher than in PTBs (P < 0.05). p53 staining was comparable in membranes from PTB and term birth pregnancies, whereas only <30% and <45% of cells were positive for p21 and p38 MAPK, respectively. In vitro cigarette smoke extract exposure increased p-p38 MAPK without any detectable change in p-p53 MAPK. Enlargement of organelles consistent with senescence phenotype was evident in pPROM and term membranes in vivo and after cigarette smoke extract treatment in vitro but was less apparent in PTBs. Histologic and biochemical resemblance of pPROM and term membranes suggests premature senescence of the membranes is a mechanistic feature in pPROM, and this can be phenocopied in an in vitro model.

Increased prevalence of preeclampsia among women undergoing procedural intervention for renal artery fibromuscular dysplasia.

BACKGROUND: Renal artery fibromuscular dysplasia (RA-FMD) has a higher prevalence among women and a presumed hormonal etiology. Although preeclampsia has a clinical presentation similar to symptomatic RA-FMD and occurs exclusively in women, associations between these 2 diseases have not been characterized. To explore epidemiologic associations between RA-FMD and preeclampsia, we administered a validated screening instrument for preeclampsia to a cohort of women with a history of pregnancy who had previously been treated with procedural intervention for symptomatic RA stenosis. METHODS: Women with a history of pregnancy who had previously undergone procedural intervention (including angioplasty and/or bypass) for symptomatic RA stenosis were identified from a prospectively maintained operative registry and screened for remote history of preeclampsia using a validated survey instrument. Univariable associations between RA-FMD and preeclampsia among participants with a history of pregnancy were evaluated using t-tests for continuous factors and chi-squared tests for dichotomous factors. Multivariable associations were evaluated using logistic regression models. RESULTS: A total of 144 women were identified who met the study inclusion criteria, including 94 with atherosclerotic RA stenosis and 50 with RA-FMD. Sixty-nine patients were contacted, 59 consented to participate, and 52 had a history of pregnancy (and therefore were at risk for preeclampsia). Participants completed the survey instrument at a mean of 7.1 ± 3.1 vs. 6.9 ± 3.6 years after RA procedural intervention, respectively. Survey responses indicated a history of preeclampsia in 19/52 (36.5%) of participants overall, including 14/27 (51.9%) with RA-FMD versus 5/20 (20.0%) with RA atherosclerosis (P = 0.02). Preeclampsia remained associated with FMD in a multivariable model adjusting for smoking status, age at time of surgery, and estimated glomerular filtration rate (odds ratio [OR] 9.51, 95% confidence interval [CI] 1.49-60.6, P = 0.017); age at the time of surgery (OR 2.78, 95% CI 1.04-7.42, P = 0.041) and estimated glomerular filtration rate (OR 3.31, 95% CI 1.29-8.52, P = 0.013) were also associated with FMD in the multivariable model. CONCLUSIONS: Women with a history of procedural intervention for symptomatic RA stenosis have an overall prevalence of preeclampsia which greatly exceeds that expected in the general population, and our results suggest that preeclampsia is specifically associated with RA-FMD. Further investigation is needed to characterize the mechanistic relationships between FMD and preeclampsia and may have potential to decrease related cardiovascular morbidity and mortality.

Endometriosis: the role of neuroangiogenesis.

Endometriosis is a common cause of pelvic pain and infertility, affecting ∼10% of reproductive-age women. Annual costs for medical and surgical care in the United States exceed $20 billion. The disorder is characterized by implants of endometrial tissue outside the uterine cavity. Endometriotic lesions induce a state of chronic peritoneal inflammation, accompanied by elevated prostaglandin, cytokine, and growth factor concentrations. The current therapy is surgical ablation of ectopic implants and hormones that block the hypothalamic-pituitary-ovarian axis, but these approaches are expensive, carry perioperative risks, or have unpleasant side effects of hypoestrogenism. Recent evidence indicates that ectopic endometriotic implants recruit their own unique neural and vascular supplies through neuroangiogenesis. It is believed that these nascent nerve fibers in endometriosis implants influence dorsal root neurons within the central nervous system, increasing pain perception in patients. We consider the mechanisms and therapeutic implications of neuroangiogenesis in these lesions and propose potential treatments for the control or elimination of endometriosis-associated pain.