Longitudinal assessment of spiral artery and intravillous arteriole blood flow and adverse pregnancy outcome.

OBJECTIVE: Superb microvascular imaging (SMI) has been shown to improve visualization of small vessels by suppressing global motions while preserving low-flow components, such as the microvessels in the placenta. We sought to determine if SMI-aided visualization of flow velocity waveforms in the spiral arteries (SA) and intravillous fetal arterioles (IVA) could predict fetal growth restriction (FGR), gestational hypertension (GH) and/or pre-eclampsia (PE). METHODS: This was a prospective longitudinal study of singleton pregnancies without fetal anomaly, receiving prenatal care in one of two medical centers over a 5-year period. Using SMI-aided color Doppler, SA and IVA flow velocity was measured at three timepoints: 11 + 0 to 14 + 0, 18 + 0 to 22 + 6 and 28 + 0 to 34 + 6 weeks of gestation. SA and IVA flow velocity waveforms were reported as resistance indices (RI). RI values were analyzed using multilevel modeling; individual regression curves were estimated and combined to obtain the reference intervals for SA-RI and IVA-RI in uncomplicated pregnancies. The primary clinical outcome was FGR and secondary outcomes were PE and GH. FGR was defined as estimated fetal weight < 10th percentile. Student's t-test was used to compare deviation from expected RI between normal and complicated pregnancies. RESULTS: Among 540 pregnancies included in the analysis, 18 (3.3%) had FGR, 31 (5.7%) PE and 61 (11.3%) GH. In uncomplicated pregnancies, the SA-RI decreased progressively with advancing gestation, whereas the IVA-RI increased with gestational age. In the third trimester, the mean SA-RI and IVA-RI values were significantly higher in the FGR group compared with pregnancies that did not develop FGR, while the mean SA-RI was significantly higher in PE compared with non-PE pregnancies. There was no significant difference in mean SA-RI or IVA-RI between pregnancies with vs those without GH at any gestational age. When all three adverse outcomes were combined, SA-RI was significantly higher in pregnancies with these outcomes when compared to uncomplicated pregnancies in the third trimester (mean ± SD, 0.29 ± 0.12 vs 0.26 ± 0.12; P = 0.02). In screening for FGR using SA-RI, the areas under the receiver-operating-characteristics curves (AUC) were 0.68, 0.73 and 0.73 in the first, second and third trimesters, respectively. The respective AUCs for IVA-RI were 0.72, 0.72 and 0.73 for each trimester. CONCLUSIONS: SA-RI and IVA-RI, measured using SMI technology, were significantly higher in pregnancies at risk for FGR in late gestation. Larger studies are needed to determine if SA and IVA flow are reliable predictors of adverse pregnancy outcome. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Risk of pre-eclampsia in patients with a maternal genetic predisposition to common medical conditions: a case-control study.

OBJECTIVE: To assess whether women with a genetic predisposition to medical conditions known to increase pre-eclampsia risk have an increased risk of pre-eclampsia in pregnancy. DESIGN: Case-control study. SETTING AND POPULATION: Pre-eclampsia cases (n = 498) and controls (n = 1864) in women of European ancestry from five US sites genotyped on a cardiovascular gene-centric array. METHODS: Significant single-nucleotide polymorphisms (SNPs) from 21 traits in seven disease categories (cardiovascular, inflammatory/autoimmune, insulin resistance, liver, obesity, renal and thrombophilia) with published genome-wide association studies (GWAS) were used to create a genetic instrument for each trait. Multivariable logistic regression was used to test the association of each continuous scaled genetic instrument with pre-eclampsia. Odds of pre-eclampsia were compared across quartiles of the genetic instrument and evaluated for significance. MAIN OUTCOME MEASURES: Genetic predisposition to medical conditions and relationship with pre-eclampsia. RESULTS: An increasing burden of risk alleles for elevated diastolic blood pressure (DBP) and increased body mass index (BMI) were associated with an increased risk of pre-eclampsia (DBP, overall OR 1.11, 95% CI 1.01-1.21, P = 0.025; BMI, OR 1.10, 95% CI 1.00-1.20, P = 0.042), whereas alleles associated with elevated alkaline phosphatase (ALP) were protective (OR 0.89, 95% CI 0.82-0.97, P = 0.008), driven primarily by pleiotropic effects of variants in the FADS gene region. The effect of DBP genetic loci was even greater in early-onset pre-eclampsia cases (at <34 weeks of gestation, OR 1.30, 95% CI 1.08-1.56, P = 0.005). For other traits, there was no evidence of an association. CONCLUSIONS: These results suggest that the underlying genetic architecture of pre-eclampsia may be shared with other disorders, specifically hypertension and obesity. TWEETABLE ABSTRACT: A genetic predisposition to increased diastolic blood pressure and obesity increases the risk of pre-eclampsia.

Chinese herbal medicine for miscarriage affects decidual micro-environment and fetal growth.

INTRODUCTION: Intrauterine growth restriction complicates 5-10% of pregnancies. This study aims to test the hypothesis that Chinese herbal formula, JLFC01, affects pregnancy and fetal development by modulating the pro-inflammatory decidual micro-environment. METHODS: Human decidua from gestational age-matched elective terminations or incomplete/missed abortion was immunostained using anti-CD68 + anti-CD86 or anti-CD163 antibodies. qRT-PCR and Luminex assay measured the effects of JLFC01 on IL-1ß- or TNF-α-induced cytokine expression in first trimester decidual cells and on an established spontaneous abortion/intrauterine growth restriction (SA/IUGR)-prone mouse placentae. The effect of JLFC01 on human endometrial endothelial cell angiogenesis was evaluated by average area, length and numbers of branching points of tube formation. Food intake, litter size, fetal weight, placental weight and resorption rate were recorded in SA/IUGR-prone mouse treated with JLFC01. qRT-PCR, Western blot and immunohistochemistry assessed the expression of mouse placental IGF-I and IGF-IR. RESULTS: In spontaneous abortion, numbers of decidual macrophages expressing CD86 and CD163 are increased and decreased, respectively. JLFC01 reduces IL-1ß- or TNF-α-induced GM-CSF, M-CSF, C-C motif ligand 2 (CCL2), interferon-γ-inducible protein-10 (IP-10), CCL5 and IL-8 production in first trimester decidual cells. JLFC01 suppresses the activity of IL-1ß- or TNF-α-treated first trimester decidual cells in enhancing macrophage-inhibited angiogenesis. In SA/IUGR-prone mice, JLFC01 increases maternal food intake, litter size, fetal and placental weight, and reduces fetal resorption rate. JLFC01 induces IGF-I and IGF-IR expression and inhibits M-CSF, CCL2, CCL5, CCL11, CCL3 and G-CSF expression in the placentae. DISCUSSION: JLFC01 improves gestation by inhibiting decidual inflammation, enhancing angiogenesis and promoting fetal growth.

Long-acting progestin-only contraceptives enhance human endometrial stromal cell expressed neuronal pentraxin-1 and reactive oxygen species to promote endothelial cell apoptosis.

CONTEXT: Despite the absence of progesterone receptor protein in human endometrial endothelial cells (HEECs), endometria of women receiving long-acting progestin-only contraceptives (LAPCs) display reduced uterine blood flow, elevated reactive oxygen species generation, increased angiogenesis, and irregularly distributed, enlarged, fragile microvessels resulting in abnormal uterine bleeding. OBJECTIVE: We propose that paracrine factors from LAPC-treated human endometrial stromal cells (HESCs) impair HEEC functions by shifting the balance between HEEC viability and death in favor of the latter. DESIGN AND SETTING: Proliferation, apoptosis, and transcriptome analyses were performed in HEECs treated with conditioned medium supernatant (CMS) derived from HESCs treated with estradiol (E2) ± medroxyprogesterone acetate or etonogestrel under normoxia or hypoxia. Mass spectrometry interrogated the CMS secretome while immunostaining for neuronal pentraxin-1 (NPTX1), cleaved caspase-3, and cytochrome c was performed in cultured HEECs and paired endometria from women using LAPCs. MAIN OUTCOME: HEEC apoptosis and its underlying mechanism. RESULTS: HESC CMS from E2 + medroxyprogesterone acetate or E2 + etonogestrel incubations under hypoxia induced HEEC apoptosis (P < .05), whereas mass spectrometry of the CMS revealed increased NPTX1 secretion (P < .05). Endothelial cleaved caspase-3 and stromal NPTX1 immunoreactivity were significantly higher in LAPC-treated endometria (P < .001). Transcriptomics revealed AKT signaling inhibition and mitochondrial dysfunction in HEECs incubated with HESC CMS. In vitro analyses proved that CMS decreased HEEC AKT phosphorylation (P < .05) and that recombinant NPTX1 (P < .05) or NPTX1 + H2O2 (P < .001) increase HEEC apoptosis and cytosolic cytochrome c levels. CONCLUSIONS: LAPC-enhanced NPTX1 secretion and reactive oxygen species generation in HESCs impair HEEC survival resulting in a loss in vascular integrity, demonstrating a novel paracrine mechanism to explain LAPC-induced abnormal uterine bleeding.

Effects of etonogestrel treatment in the reproductive organs and uterine arteries of nonoophorectomized guinea pigs.

The endometria of women treated with long-term progestin-only contraceptives (LTPOCs) display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow, oxidative stress, and unpredictable focal abnormal endometrial bleeding. Because human studies on the effects of LTPOC treatment are constrained for ethical and practical reasons, we assessed the suitability of nonoophorectomized guinea pigs (GPs) to best mimic the hormonal milieu of women. The present study demonstrates that treatment of GPs parallels the morphological changes following LTPOC treatment of the human endometrium and ovaries. Specifically, treatment resulted in larger hyperemic, uteri compared with controls. Histopathologic and immunohistochemical analysis demonstrated fewer endometrial glands, decreased luminal mucus, increased numbers of blood vessels, and focal hemorrhage. While increased staining for the cell mitosis marker, Ki67, was present in the zona functionalis, no such increase occurred in the basalis. Lastly, effects on vasomotor features of uterine arteries suggest changes that favor increased resistance and reduced blood flow promoting decreased ability to withstand elevations in transmural pressure.

Pro-inflammatory cytokine-stimulated first trimester decidual cells enhance macrophage-induced apoptosis of extravillous trophoblasts.

OBJECTIVE: As human blastocyst-derived extravillous trophoblasts (EVTs) invade the early decidua, they are positioned to interact with immune cells and resident decidual cells, and remodel spiral arteries into high capacity vessels that increase blood flow to the developing fetal-placental unit. Shallow EVT invasion elicits incomplete vascular transformation and reduces uteroplacental blood flow that presages adverse pregnancy outcomes. Excess macrophages in the decidua induce EVT apoptosis via tumor necrosis factor-alpha (TNF-α) secretion. Our previous observation that pro-inflammatory cytokines enhance neutrophil and macrophage activator granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in first trimester decidual cells is now extended to include: (1) the specific macrophage activator M-CSF; (2) macrophage activation and subsequent enhancement of EVT apoptosis by both GM-CSF and M-CSF. STUDY DESIGN: Quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay assessed M-CSF expression in first trimester decidual cells incubated with interleukin-1 beta (IL-1ß) or TNF-α. Peripheral monocyte-derived macrophages pre-incubated with conditioned media from decidual cell cultures were co-cultured with a first trimester EVT cell line, HTR-8/SVneo cells. Macrophage activation was examined and EVT apoptosis evaluated by DNA fragmentation, caspase activation and cell membrane asymmetry. RESULTS: IL-1ß or TNF-α significantly enhanced M-CSF expression in first trimester decidual cells. The conditioned media from these cultures activates macrophages, which promote caspase 3/7-dependent EVT apoptosis with antibodies against GM-CSF or M-CSF blocking this effect. CONCLUSIONS: Pro-inflammatory cytokines increases synthesis of M-CSF in first trimester decidual cells. Both GM-CSF and M-CSF activate macrophages, which initiate caspase-dependent EVT apoptosis.

The effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion membrane, in vitro.

Abruption-induced thrombin generation and inflammation/infection induced cytokine production have both been associated with fetal membrane (FM) weakening and preterm premature rupture of the fetal membranes (PPROM). Using our in vitro model system we have demonstrated that thrombin, and separately the cytokines, tumor necrosis factor-alpha (TNFα) and interleukin-1-beta (IL-1ß), remodel and weaken full thickness FM. Additionally, we have reported that the anti-oxidant and NFκB inhibitor, alpha-lipoic acid (LA), blocks these thrombin and cytokine induced effects. The purpose of these studies was to determine whether thrombin and cytokines directly weaken the amnion membrane (AM), the major load-bearing component of FM. Isolated AM or full thickness FM fragments from unlabored Cesarean deliveries were incubated with thrombin, TNFα, or IL-1ß, for 48 h. Rupture strength (breaking force) of each fragment was thereafter determined using our published methodology. Biochemical evidence of remodeling and apoptosis; immunoreactive Matrix Metalloproteinase 9 (MMP9), Tissue Inhibitor of Matrix Metalloproteinase 3 (TIMP3) and cleaved poly (ADP-ribose) polymerase (C-PARP) levels in tissue extracts, were determined by western blot and densitometry. Thrombin induced a dose-dependent weakening of isolated AM (P < 0.001) coupled with dose dependent increases in PARP cleavage, and reciprocal increases and decreases, respectively, in MMP9 and TIMP3 protein (all P < 0.01). Thrombin receptor activating peptide-6 (TRAP) also weakened isolated AM. Neither TNFα nor IL-1ß weakened isolated AM. However, both cytokines weakened AM when it was incubated together with the choriodecidua as part of full thickness FM (P < 0.001). Cytokine-conditioned choriodecidua medium also weakened isolated AM (P < 0.001). Under conditions in which cytokines weakened the AM, the changes in MMP9, TIMP3 and PARP cleavage were consistent with those seen after thrombin incubation. LA blocked the FM weakening and remodeling effects. In summary, thrombin weakens AM directly whereas cytokines weaken AM indirectly by causing the release of soluble intermediates from the choriodecidua.

Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro.

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1ß, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption.

hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor for immunotherapy of uterine serous papillary carcinoma.

BACKGROUND: Uterine serous papillary adenocarcinoma (USPC) is a highly aggressive variant of endometrial cancer. Human immuno-conjugate molecule (hI-con1) is an antibody-like molecule targeted against tissue factor (TF), composed of two human Factor VII (fVII) as the targeting domain, fused to human immunoglobulin (Ig) G1 Fc as an effector domain. We evaluated hI-con1 potential activity against primary chemotherapy-resistant USPC cell lines expressing different levels of TF. METHODS: A total of 16 formalin-fixed, paraffin-embedded USPC samples were evaluated by immunohistochemistry (IHC) for TF expression. Six primary USPC cell lines, half of which overexpress the epidermal growth factor type II (HER2/neu) receptor at 3+ levels, were assessed by flow cytometry and real-time PCR for TF expression. Sensitivity to hI-con1-dependent cell-mediated cytotoxicity (IDCC) was evaluated in 5-hour-chromium release assays. Finally, to investigate the effect of interleukin-2 (IL-2) on IDCC, 5-h (51)Cr assays were also conducted in the presence of low doses of IL-2 (i.e., 50-100 IU ml(-1)). RESULTS: Cytoplasmic and/or membrane TF expression was observed in all 16 (100%) USPC samples tested by IHC, but not in normal endometrium. High expression of TF was found in 50% (three out of six) of the USPC cell lines tested by real-time PCR and flow cytometry when compared with normal endometrial cells (NECs; P<0.001). Uterine serous papillary adenocarcinoma cell lines overexpressing TF, regardless of their high or low HER2/neu expression, were highly sensitive to IDCC (mean killing+/-s.d., 65.6+/-3.7%, range 57.5-77.0%, P<0.001), although negligible cytotoxicity against USPC was seen in the absence of hI-con1 or in the presence of Rituximab control antibody. The addition of low doses of IL-2 further increased the cytotoxic effect induced by hI-con1 against chemotherapy-resistant USPC. CONCLUSION: hI-con1 induces strong cytotoxicity against primary chemotherapy-resistant USPC cell lines overexpressing TF. The hI-con1 may represent a novel therapeutic agent for the treatment of patients harbouring advanced, recurrent and/or metastatic USPC refractory to standard treatment modalities.

Human labor is associated with reduced decidual cell expression of progesterone, but not glucocorticoid, receptors.

CONTEXT: Unchanging plasma progesterone (P4) levels suggest that human labor is initiated by reduced P4 receptor (PR) expression, which elicits functional P4 withdrawal. The glucocorticoid receptor (GR) is also implicated in this process. OBJECTIVE: Our objective was to compare PR and GR staining in human decidual cells (DCs) and interstitial trophoblasts (ITs) of gestational age-matched pre- and postcontraction specimens and to evaluate steroid effects on PR and GR expression in human DC cultures. INTERVENTIONS AND MAIN OUTCOME MEASURES: Decidua basalis and parietalis sections were immunostained for PR or GR and then for the cytoplasmic DC and IT markers vimentin and cytokeratin. Western blotting measured PR and GR levels in nuclear extracts of cultured leukocyte-free term DCs after incubation with estradiol-17beta (E2) with or without medroxyprogesterone acetate (MPA). RESULTS: PR histological scores (HSCOREs) were significantly higher in DC nuclei from pre- vs. post-uterine-contraction decidua basalis and parietalis sections with PR immunostaining absent from ITs. In contrast, immunoreactive GR was localized in IT and DC nuclei. GR HSCORES were significantly higher in ITs than DCs but similar in pre- vs. post-uterine-contraction specimens. In term DC monolayers, PR-A and PR-B were enhanced by E2 and inhibited by MPA, whereas E2 plus MPA produced intermediate PR expression. The GR was constitutively expressed. CONCLUSIONS: In post- vs. pre-uterine-contraction specimens, significantly lower HSCOREs in DC nuclei, but not IT, and unchanging GR levels in DCs and ITs suggest that functional P4 withdrawal may occur in DCs and is unlikely to involve the GR. Nuclear extracts from DC monolayer cultures express steroid-regulated PR-A and PR-B and constitutive GR.

Involvement of human decidual cell-expressed tissue factor in uterine hemostasis and abruption.

Vascular injury increases access and binding of plasma-derived factor VII to perivascular cell membrane-bound tissue factor (TF). The resulting TF/VIIa complex promotes hemostasis by cleaving pro-thrombin to thrombin leading to the fibrin clot. In human pregnancy, decidual cell-expressed TF prevents decidual hemorrhage (abruption). During placentation, trophoblasts remodel decidual spiral arteries into high conductance vessels. Shallow trophoblast invasion impedes decidual vascular conversion, producing an inadequate uteroplacental blood flow that elicits abruption-related placental ischemia. Thrombin induces several biological effects via cell surface protease activated receptors. In first trimester human DCs thrombin increases synthesis of sFlt-1, which elicits placental ischemia by impeding angiogenesis-related decidual vascular remodeling. During pregnacy, the fibrillar collagen-rich amnion and choriodecidua extracellular matrix (ECM) provides greater than additive tensile strength and structural integrity. Thrombin acts as an autocrine/paracrine mediator that degrades these ECMs by augmenting decidual cell expression of: 1) matrix metalloproteinases and 2) interleukin-8, a key mediator of abruption-associated decidual infiltration of neutrophils, which express several ECM degrading proteases. Among the cell types at the maternal fetal interface at term, TF expression is highest in decidual cells indicating that this TF meets the hemostatic demands of labor and delivery. TF expression in cultured term decidual cells is enhanced by progestin and thrombin suggesting that the maintenance of elevated circulating progesterone provides hemostatic protection and that abruption-generated thrombin acts in an autocrine/paracrine fashion on decidual cells to promote hemostasis via enhanced TF expression.

Progestin and thrombin regulate tissue factor expression in human term decidual cells.

CONTEXT: Perivascular cell membrane-bound tissue factor (TF) initiates hemostasis via thrombin generation. The identity and potential regulation of TF-expressing cells at the human maternal-fetal interface that confers hemostatic protection during normal and preterm delivery is unclear. OBJECTIVES: The objective of the study were to identify TF-expressing cells at the maternal-fetal interface in term and preterm decidual sections by immunohistochemistry and evaluate progestin, thrombin, TNF-alpha, and IL-1beta effects on TF expression by cultured human term decidual cells (DCs). INTERVENTIONS AND MAIN OUTCOME MEASURES: Serial placental sections were immunostained for TF. Leukocyte-free term DC monolayers were incubated with 10(-8) M estradiol (E2) or E2 plus 10(-7) M medroxyprogestrone acetate (MPA) +/- thrombin or TNF-alpha or IL-1beta. ELISA and Western blotting assessed TF in cell lysates. Quantitative real-time RT-PCR measured TF mRNA levels. RESULTS: Immunolocalized TF in DC membranes in preterm and term placental sections displayed higher Histologic Scores than villous mesenchymal cells (P < 0.05). TF was undetected in interstitial or extravillous trophoblasts. Compared with DCs incubated with E2, MPA and 2.5 U/ml thrombin each doubled TF levels (P < 0.05) and E2 + MPA + thrombin further doubled TF levels (P < 0.05), whereas TNF-alpha and IL-1beta were ineffective. Western blotting confirmed the ELISA results. Quantitative RT-PCR revealed corresponding changes in TF mRNA levels. CONCLUSIONS: In human term placental sections, DC-expressed TF exceeds that of other cell types at the maternal-fetal interface and is localized at the cell membranes in which it can bind to factor VII and meet the hemostatic demands of labor and delivery via thrombin formation. Unlike the general concept that TF is constitutive in cells that highly express it, MPA and thrombin significantly enhanced TF expression in term DC monolayers.

Liver dysfunction in pregnancy: an important cause of maternal and perinatal morbidity and mortality in Pakistan.

The objective of this study was to evaluate the maternal and perinatal outcome of women with liver dysfunction during pregnancy. The study involved a prospective observational study design and was carried out at the Dow University of Health Sciences and Civil Hospital Karachi, Pakistan. A total of 800 women, who delivered during the study period from January 2006 to September 2006, constituted the study population. Pregnant women with liver dysfunction underwent evaluation for the aetiology of their liver dysfunction, including screening for hepatitis E. Thirty-five women were identified with liver dysfunction. Fourteen (40%) presented in the second trimester and 21 (60%) presented in the third trimester. Twenty-two of the 35 women (63%) had isolated acute hepatitis E; five (14%) had HELLP (haemolysis, elevated liver enzymes and low platelet count) syndrome; two (6%) had intrahepatic cholestasis of pregnancy (IHCP), two had acute fatty liver of pregnancy (AFLP) and two women had hepatitis A. A specific diagnosis was not reached in two women who died prior to delivery. In women with hepatitis E, the mean values of bilirubin and alanine transaminase were 12 mg/dL and 675 U/L, respectively. Abnormal coagulation parameters were present in 20 (57%) of the women and in 18 of 22 (82%) with hepatitis E. Fulminant hepatic failure (FHF) was seen in four patients. Seven women (20%) underwent caesarean section, 26 (74%) delivered vaginally and two women died undelivered. There were six maternal deaths in the study population; two were due to hepatitis E, one each from HELLP and AFLP, and two remained undiagnosed. The overall perinatal mortality within the group was 43%. Hepatitis E was the most common cause of FHF and maternal death in pregnant women with liver dysfunction.

Pre-eclampsia is associated with dendritic cell recruitment into the uterine decidua.

Pre-eclampsia is a leading cause of fetal and maternal morbidity and mortality that preferentially affects primiparous patients. It is associated with systemic inflammation and impaired trophoblast invasion of the decidua. Decidual cells are the major cell type of the pregnant endometrium. Macrophages and dendritic cells are major specialized antigen-presenting cells that promote both innate immunity and immune tolerance. Macrophage infiltration is implicated in impaired trophoblast invasion that leads to pre-eclampsia. By contrast, the potential modulating role of decidual dendritic cells in the genesis of pre-eclampsia has not been investigated. Interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, has been implicated in the genesis of pre-eclampsia. Thus, we postulate that pre-eclampsia would be associated with enhanced decidual dendritic cells infiltration and that IL-1beta would enhance the production of relevant dendritic cell-recruiting chemokines. We used immunohistochemistry to demonstrate a marked infiltrate of immature and mature dendritic cells in pre-eclamptic decidua. Further, immunohistochemistry and immunoassays of placental bed biopsies revealed that pre-eclamptic decidua displays elevated levels of several monocyte- and dendritic cell-recruiting chemokines. Leukocyte-free first-trimester decidual cells were then treated with IL-1beta, which enhanced the mRNA and protein expression of these chemokines. The current study also confirmed previous reports that macrophages directly impaired trophoblast invasion and that this inhibitory effect is augmented by the conditioned medium of IL-1beta-treated first-trimester decidual cells. However, unlike macrophages, dendritic cells did not directly impede trophoblast invasion. This study demonstrates that the inflammatory milieu of pre-eclampsia induces decidual cells to promote dendritic cell infiltration. Given their unusual versatility in mediating both immunity and tolerance, these novel findings suggest that dendritic cells may play a critical role either in the pathogenesis of pre-eclampsia or its prevention in subsequent pregnancies.

Expression of Toll-like receptors in the human decidua.

BACKGROUND: Successful trophoblast invasion and transformation of the maternal spiral arteries requires that the pregnant endometrium (i.e., decidua) act in an immunologically paradoxical fashion, accepting the semi-allogenic placenta, while maintaining host defenses against an array of microbial pathogens. In contrast to the growing evidence that the immune surveillance molecules known as Toll-like receptors (TLRs) are expressed by trophoblasts and fetal membranes, to date, no studies have been conducted on the decidua. METHODS: Decidual tissues and cells were obtained from women undergoing first trimester elective terminations or repeat Cesarean sections and analyzed at both the protein and mRNA level. RESULTS: We now demonstrate for the first time that human decidua differentially express TLRs and their downstream signaling molecules as well as TLR stimulated induction of cytokine production in the first and third trimester of pregnancy. CONCLUSIONS: These findings suggest that the decidua is a critical component of the innate immune response in pregnancy. Moreover, the results have implications for the success or failure of compromised pregnancies in early or late gestation.

Long-term progestin-only contraceptives result in reduced endometrial blood flow and oxidative stress.

CONTEXT: Because of their safety and efficacy, long-term progestin-only contraceptives (LTPOCs) are well-suited for women with restricted access to health care. However, abnormal uterine bleeding (AUB) causes half of all users to discontinue therapy within 12 months. Endometria of LTPOC-treated patients display aberrant angiogenesis with abnormally enlarged, thin-walled, fragile blood vessels, inflammation, and focal hemorrhage. In this study, similar effects were observed with a new third-generation implantable LTPOC. OBJECTIVE: We hypothesized that LTPOC reduces uterine and endometrial blood flow, leading to hypoxia/reperfusion, which triggers the generation of reactive oxygen species. The latter induce aberrant angiogenesis, causing AUB. DESIGN: Endometrial perfusion was measured by laser-Doppler fluxmetry in women requesting LTPOCs. Endometrial biopsies were obtained for in vivo and in vitro experiments. SETTING: The study was conducted in the Yale University School of Medicine and Family-Planning Center in Western Australia. PATIENTS: Seven women 18 yr or older requesting implantable LTPOCs were recruited in Western Australia. INTERVENTION: Women received etonorgestrel implants. MAIN OUTCOME: LTPOC treatment resulted in reduced endometrial perfusion and increased endometrial oxidative damage. CONCLUSIONS: We propose that LTPOCs result in hypoxia reperfusion, which leads to aberrant angiogenesis resulting in AUB.

Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells.

OBJECTIVE: Chemokines initiate the immune response by controlling leukocyte migration and lymphocyte development. Macrophage infiltration of the decidua has been implicated in the genesis of recurrent miscarriage and preeclampsia. Therefore, we determined whether cultured human decidual cells produce monocyte/macrophage-recruiting chemokines in response to a potent pro-inflammatory cytokine, interleukin-1beta (IL-1beta), and whether decidual cell-conditioned medium contains monocyte- and macrophage-chemoattractant activity. METHODS: Leukocyte-free first trimester decidual cells were treated for 6h with estradiol (E(2)) and medroxyprogesterone acetate (MPA) to mimic the steroidal milieu of pregnancy, or E(2) and MPA and IL-1beta (1 ng/ml) to mimic inflamed decidua. Total RNA was used for cDNA synthesis. Biotinylated cRNAs were generated and chemically fragmented for hybridization on Affymetrix HG_U133 Plus 2.0 chips followed by fluorescence labeling and optical scanning. Raw data generated from Affymetrix GCOS 1.2 (GeneChip Operating Software) were analyzed by GeneSpring 7.2 software. Subsequently microarray results were validated by real time RT-PCR and Western blotting. A functional study of monocyte migration was carried out also using conditioned media from culture. RESULTS: Five chemokines responsible for monocyte/macrophage chemoattraction and activation, including C-C motif ligand 2 (CCL2), CCL5, C-X-C motif ligand 2 (CXCL2), CXCL3 and CXCL8, were markedly elevated from 29- to 975-fold after exposure to IL-1beta in cultured first trimester decidual cells. The results of real-time RT-PCR (up-regulation from 43- to 3069-fold) and Western blotting (up-regulation from 15- to 300-fold) confirmed the microarray findings. Monocyte migration was significantly induced by the conditioned medium from IL-1beta-treated decidual cells. CONCLUSIONS: Treatment of first trimester decidual cells with IL-1beta induces secretion of monocyte/macrophage recruiting-chemokines and promotes monocyte migration. Extrapolation of these in vitro results to the milieu of implantation site suggests a mechanism whereby IL-1beta could mediate excessive macrophage infiltration of the decidua.

Role of cytokines in preterm labor and birth.

Preterm birth (defined as delivery prior to 37 weeks' gestation) complicates 5-10% of all births. It is a major cause of perinatal mortality and morbidity. Approximately 20% of all preterm births are iatrogenic resulting from obstetric intervention for maternal and/or fetal indications. Of the remainder, 2/3 are spontaneous preterm labor with or without preterm premature rupture of the membranes (pPROM). Preterm labor is a syndrome rather than a diagnosis since the etiologies are varied. Risk factors include, among others, pPROM, cervical insufficiency, pathologic uterine distention (polyhydramnios, multiple gestation), uterine anomalies, intrauterine infection/inflammation, and social factors (stress, smoking, heavy work). The final common pathway appears to be activation of the inflammatory cascade. Bacterial colonization and/or inflammation of the choriodecidual interface induces production of pro-inflammatory cytokines that, in turn, lead to neutrophil activation and the synthesis and release of uterotonins such as prostaglandins (which cause uterine contractions) and metalloproteinases (that weaken fetal membranes and remodel cervical collagen). This monograph reviews the role of cytokines in the pathophysiology of preterm labor and delivery.

Metalloproteinase expression by control and telomerase immortalized human endometrial endothelial cells.

Vascular endothelial cells play a critical role in the maintenance of endometrial homeostasis. Indeed many pathological conditions causing abnormal endometrial bleeding including progestin only contraception, hormone replacement therapy, endometrial polyps, myomas, hyperplasia and cancer are associated with aberrant angiogenesis. Critical to the process of angiogenesis is the breakdown of the surrounding tissues by matrix metalloproteases (MMPs). In addition to the cells surrounding the endometrial endothelial cells, the endothelial cells themselves produce their own panel of MMPs. We now characterize the specific MMPs that are expressed by endothelial cells derived from human endometrium. These include MMP-1, MMP-2 and MMP-10 but not MMP-3. In addition, in order to successfully carry out consistent, homogeneous and sufficient numbers of studies we investigated the in vitro expression of the MMPs with both freshly isolated, early passaged endometrial endothelial cells (HEECs) as well as with newly telomerase immortalized HEECs (T-HEECs). The latter were karyotypically normal and expressed classic endothelial cell endpoints such as tubulogenesis on matrigel and expression of the endothelial cell markers CD-31 (PECAM), von Willebrand's factor, and the Tie-2 receptors. The levels of MMP expression as well as that of the metalloprotease inhibitors TIMP-1 and TIMP-2 were similar in parent and immortalized endothelial cells.

Protein Z, protein S levels are lower in patients with thrombophilia and subsequent pregnancy complications.

OBJECTIVE: We posit that low levels of protein S (PS) and protein Z (PZ) contribute to adverse pregnancy outcome (APO). PATIENTS: We evaluated 103 women with subsequent normal pregnancy outcome (NPO), 106 women with APO, and 20 women with thrombophilia (TP). METHODS: We compared first trimester (1st TRI) PZ levels in 103 women with NPO, 106 women with APO, and in 20 women with TP. We compared plasma levels of PZ and free PS antigen during the second (2nd TRI) and third trimesters (3rd TRI) of pregnancy in 51 women with APO and 51 matched women with NPO. RESULTS: The mean 1st TRI PZ level was significantly lower among patients with APO, compared to pregnant controls (1.81 +/- 0.7 vs. 2.21 +/- 0.8 microg mL(-1), respectively, P < 0.001). Of patients with known TP, those with APO had a tendency for lower mean PZ levels compared to those TP women with NPO (1.5 +/- 0.6 vs. 2.3 +/- 0.9 microg mL(-1), respectively, P < 0.0631). There was a significant decrease in the PZ levels in patients with APO compared to NPO (2nd TRI 1.5 +/- 0.4 vs. 2.0 +/- 0.5 microg mL(-1), P < 0.0001; and 3rd TRI 1.6 +/- 0.5 vs. 1.9 +/- 0.5 microg mL(-1), P < 0.0002). Protein S levels were significantly lower in the 2nd and 3rd TRIs among patients with APO compared to patients with NPO (2nd TRI 34.4 +/- 11.8% vs. 38.9 +/- 10.3%, P < 0.05, respectively; and 3rd TRI 27.5 +/- 8.4 vs. 31.2 +/- 7.4, P < 0.025, respectively). CONCLUSIONS: We posit that decreased PZ and PS levels are additional risk factors for APO.